Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405413200 on June 16, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35341-35352, August 20, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/34/35341    most recent
M405413200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tikunova, S. B.
Right arrow Articles by Davis, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tikunova, S. B.
Right arrow Articles by Davis, J. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C*

Svetlana B. Tikunova{ddagger} and Jonathan P. Davis

From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210

Received for publication, May 14, 2004 , and in revised form, June 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin CF27W. Trp27 accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin CF27W. To sensitize the N-domain of cardiac troponin CF27W to calcium, we individually substituted the hydrophobic residues Phe20, Val44, Met45, Leu48, and Met81 with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin CF27W ~2.1–15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6–8.7-fold faster) rather than to slower calcium dissociation rates (1.2–2.9-fold slower). The regulatory N-domains of cardiac troponin CF27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2–2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Striated muscle contraction is initiated by Ca2+ binding to the troponin (Tn)1 complex, which consists of three subunits: TnC (the Ca2+-binding subunit), TnI (the inhibitory subunit), and troponin T (the tropomyosin-binding subunit) (for review, see Refs. 1 and 2). TnC belongs to the EF-hand superfamily of Ca2+-binding proteins (for review, see Ref. 3) and consists of N- and C-terminal globular domains, connected by a flexible central {alpha}-helix (for review, see Ref. 4). Each globular domain possesses a pair of EF-hand helix-loop-helix Ca2+-binding motifs. The EF-hands are numbered I–IV, and the helices flanking the loops are labeled A–H, with an additional ~14-residue N-helix, absent in the closely related EF-hand Ca2+-binding protein, calmodulin (CaM).

Numerous studies have concentrated on the metal binding properties of TnC. Two isoforms of TnC exist in vertebrates, one found in fast skeletal muscle (sTnC) and the other in slow skeletal and cardiac muscle (cTnC). Each domain of sTnC is capable of binding two Ca2+ ions, with the two C-domain EF-hands possessing dramatically higher Ca2+ affinity and slower Ca2+ exchange rates than the two N-domain EF-hands (5, 6). Both domains of sTnC are also capable of binding Mg2+ competitively and with physiologically relevant affinities (7, 8). Because of their high Ca2+ and Mg2+ affinities and slow exchange rates, the C-domain sites are occupied by either Ca2+ or Mg2+ even under resting physiological conditions and thus are generally believed to play a structural role by anchoring sTnC into the sTn complex. On the other hand, the N-domain sites of sTnC are believed to play a direct role in the regulation of muscle contraction and relaxation (for review, see Ref. 4). The Ca2+ and Mg2+ binding properties of the structural C-domain sites of cTnC are similar to those of sTnC (9). However, the regulatory N-domain of cTnC is capable of binding only one Ca2+ ion through its second EF-hand. The first EF-hand of cTnC is unable to bind Ca2+ because of a single residue insertion (Val28) and two chelating residue substitutions (Asp29 -> Leu and Asp31 -> Ala) (for review, see Ref. 10).

NMR and x-ray studies of sTnC demonstrated that Ca2+ binding to the N-domain sites causes a large conformational transition from a closed to an open state, in which helices B and C (BC unit) move away from helices N, A, and D (NAD unit) (11–13). This reorientation of the helices leads to the exposure of a hydrophobic patch on the surface of the protein, allowing the interactions of the N-domain of sTnC with the C-domain of sTnI, initiating the cascade of events that ultimately leads to muscle contraction (for review, see Ref. 4). In contrast, Ca2+ binding to the regulatory site of cTnC does not cause a large structural transition, with the N-domain remaining essentially in a closed state (14, 15), at least in part because of the defunct first EF-hand. Generally, Ca2+ sensor proteins, such as sTnC and CaM, undergo large conformational transitions upon Ca2+ binding, which allows them to interact with their target proteins or enzymes to transduce Ca2+ signals into a variety of cellular responses. On the other hand, Ca2+ buffer proteins, such as parvalbumin and calbindin D9K, undergo only minor conformational changes upon Ca2+ binding and are involved in Ca2+ buffering or transport (for review, see Ref. 16). Thus, cTnC is an unusual Ca2+ sensor, because Ca2+ binding alone to its regulatory N-domain does not cause large structural rearrangements. However, binding of the cardiac TnI peptide (encompassing residues 147–163) leads to the structural opening of the Ca2+-bound N-domain of cTnC (17).

Congestive heart failure can be associated with desensitization of the myocardium to Ca2+ and depressed cardiac contractility (for review, see Ref. 18). Drugs known as "Ca2+ sensitizers" increase the Ca2+ sensitivity of the myocardial contractile apparatus without elevation of intracellular Ca2+ (for review, see Refs. 19–21). Because cTnC plays an essential role in the regulation of cardiac muscle mechanics, it represents an attractive target for therapeutic Ca2+-sensitizing compounds. However, there are no available drugs that selectively bind cTnC and increase its Ca2+ binding affinity. Ideally, Ca2+-sensitizing agents should not decrease the rate of Ca2+ dissociation from the regulatory domain of cTnC, because impaired relaxation would be an undesirable consequence of Ca2+ sensitization. Understanding the factors controlling Ca2+ binding and exchange with cTnC could lead to the rational design of drugs useful in the treatment of heart disease.

The main objective of this work was to design mutations that sensitize the regulatory N-domain of cTnC to Ca2+. Previous studies demonstrated that mutation of a single hydrophobic residue, which does not directly ligate Ca2+, can either dramatically increase or decrease Ca2+ affinity, depending on the location of the residue within the tertiary structure of the EF-hand protein (22–25). Ca2+ binding to the second regulatory EF-hand of cTnC, its mutants, and its isoforms has been studied using the Phe27 -> Trp substitution immediately preceding the first defunct Ca2+-binding loop (26–29). We have utilized this substitution and introduced five additional individual mutations (based on their location within the tertiary structure of the protein) designed to increase the Ca2+ sensitivity of the regulatory domain of cTnC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Phenyl-Sepharose CL-4B and EGTA were purchased from Sigma. Quin-2 was purchased from Calbiochem (La Jolla, CA). All other chemicals were of analytical grade.

Protein Mutagenesis and Purification—The human cTnC pET3a plasmid was a generous gift from Dr. Lawrence B. Smillie (University of Alberta, Edmonton, Canada). cTnCF27W and its mutants were constructed from the cTnC plasmid by primer-based site-directed mutagenesis using Stratagene (La Jolla, CA) QuikChange site-directed mutagenesis kit. All of the mutations were confirmed by DNA sequence analysis. The plasmids for cTnCF27W and mutants were transformed into Escherichia coli BL21(DE3)pLysS cells (Novagen). Expression and purification of cTnCF27W and mutants were carried out as previously described for sTnC (25). The purified cTnCF27W and mutants were dialyzed against three exchanges of 4 liters of 10 mM MOPS, 90 mM KCl, 0.5 mM DTT, pH 7.0, at 4 °C. Protein concentration was calculated by absorbance at 280 nm using an extinction coefficient of 9770 cm–1 M–1. The purity of the proteins was checked by SDS-PAGE and was determined to be 98 ± 1%, using Labworks 4.0 Software (UVP Bioimaging Systems, St. Louis Park, MN).

Determination of Ca2+ Affinities—All of the steady state fluorescence measurements were performed using a PerkinElmer LS5 spectrofluorimeter at 15 °C. Trp fluorescence was excited at 285 nm and monitored at 345 nm as microliter amounts of CaCl2 were added to 1 ml of each cTnCF27W mutant (0.6 µM) in 200 mM MOPS (to prevent pH changes upon the addition of Ca2+), 90 mM KCl, 2 mM EGTA, 1 mM DTT, pH 7.0, in the absence or presence of 3 mM MgCl2, at 15 °C. The [Ca2+]free was calculated using the computer program EGCA02 developed by Robertson and Potter (30). The Ca2+ affinities were reported as a dissociation constant, Kd(Ca), representing a mean of at least three separate titrations ± S.D. The data were fit with a logistic sigmoid function (mathematically equivalent to the Hill equation), as previously described (25).

Determination of Mg2+ Affinities—Two methods were utilized to determine the Mg2+ binding affinities of the regulatory domain of cTnCF27W and its mutants. In the first method, Mg2+ binding affinities (Kd(Mg)) were calculated from a decrease in the apparent Ca2+ affinities caused by 3 mM Mg2+, using the equation Kd(Mg) = [Mg2+]/(Kd(Caapp)/Kd(Ca) – 1), where Kd(Ca) and Kd(Caapp) are the Ca2+ affinities in the absence and presence of 3 mM Mg2+, respectively (determined as described above). In the second method, Trp fluorescence was excited at 285 nm and monitored at 345 nm as microliter amounts of MgCl2 were added to 1 ml of each cTnCF27W mutant (0.6 µM) in 200 mM MOPS, 90 mM KCl, 2 mM EGTA, 1 mM DTT, pH 7.0, and 3.98–10 µM [Ca2+]free (i.e. [Ca2+] required to achieve at least 60% saturation of the N-domain of each individual protein) at 15 °C. The Mg2+ affinities were calculated using the equation Kd(Mg) = Kd(Mgapp)/([Ca2+]free/Kd(Ca) + 1), where Kd(Mgapp) is the negative logarithm of [Mg2+]free producing half-maximal fluorescence.

Determination of Ca2+ Dissociation Rates—Ca2+ dissociation rates (koff(Ca)) were measured using an Applied Photophysics Ltd. (Leather-head, UK) model SX.18 MV stopped-flow instrument with a dead time of 1.6 ms at 15 °C. The samples were excited using a 150-W xenon arc source. The Trp emission was monitored through a UV-transmitting black glass filter (UG1) from Oriel (Stratford, CT). The data were fit using a program (by P. J. King, Applied Photophysics Ltd.) that utilizes the nonlinear Levenberg-Marquardt algorithm. Each koff(Ca) value represents an average of at least three separate experiments, each averaging at least five traces fit with a single exponential equation (variance < 8 x 10–4). koff(Ca) was also directly measured using the fluorescent Ca2+ chelator Quin-2. Quin-2 was excited at 330 nm with its emission monitored through a 510-nm broad band pass interference filter (Oriel (Stratford, CT)). Each koff(Ca) value represents an average of at least three separate experiments, each averaging at least five traces fit with a single or double exponential equation, depending on the time frame of data collection, because Quin-2 reports Ca2+ dissociation rates from both the N-and C-domain of cTnCF27W proteins (variance < 9 x 10–5). Fitting the data with a single or double exponential equation gave similar results for the rate of Ca2+ dissociation from the N-domain of cTnCF27W proteins within appropriately chosen time frames. The buffer used in the stopped-flow experiments was 10 mM MOPS, 90 mM KCl, 1 mM DTT, pH 7.0, in the absence or presence of 3 mM MgCl2.

Calculation of Ca2+ Association Rates—The Ca2+ association rates (kon(Ca)) were calculated using the relationship kon(Ca) = koff(Ca)/Kd(Ca), where koff(Ca) represents the release of a single Ca2+ ion, and Kd(Ca) represents the binding event of a single Ca2+ ion to the N-domain of cTnC, as previously described (25).

Exposing cTnCF27W and Its Mutants to Artificial Ca2+ Transients— ACTs of various amplitudes and durations can be generated in a stopped-flow apparatus by rapidly mixing one solution containing a known concentration of Ca2+ against another solution containing a known concentration of Ca2+ chelator (31). This technique can be used to estimate the Ca2+ association rate to Ca2+-binding proteins/enzymes (31). The buffer used in the ACT experiments was 10 mM MOPS, 90 mM KCl, 1 mM DTT, 3 mM MgCl2, pH 7.0, at 15 °C. ACTs were generated by rapidly mixing each cTnCF27W protein (2 µM) + 1 mM EGTA in buffer against buffer with increasing [Ca2+]. As the [Ca2+] was increased from 5 to 750 µM (before mixing), the duration of the ACTs increased from 0.7 to 1.0 ms (as determined by computer simulations described below), whereas the amplitude (one-half of [Ca2+] before mixing) of the ACTs increased 150-fold. Before mixing, the N-domain of each cTnCF27W protein was in the apo state. After mixing, the N-domain of each cTnCF27W protein was transiently occupied by Ca2+, prior to EGTA chelating [Ca2+]free and removing Ca2+ from the protein. Because the Ca2+ association event (indicated by the increase in Trp fluorescence) occurred primarily during the mixing time of the instrument, only the Ca2+ dissociation event was visualized (as the decrease in Trp fluorescence) for each [Ca2+] that did not saturate EGTA and the protein. 0% occupancy was determined by mixing each cTnCF27W protein (2 µM) + 1 mM EGTA in buffer against buffer without added Ca2+, whereas 100% occupancy was determined by mixing each cTnCF27W protein (2 µM) + 1 mM EGTA against buffer with added [Ca2+] that exceeded the [EGTA] and fully saturated the protein. For every ACT, the percentage of transient occupancy for each cTnCF27W protein was determined at a selected time point after mixing was complete (typically ≥2 ms). Each transient occupancy trace represents the mean of three separate experiments (averaging at least 10 traces each), with the percentage of occupancy (at the selected time point) reported as a mean ± S.D.

Computer Modeling—To estimate the Ca2+ association rate to the N-domain of cTnCF27W and its mutants, computer simulations were performed using KSIM version 1.1 (N. C. Millar, UCLA School of Medicine, Los Angeles, CA) (32), which solved the set of chemical equations listed below numerically using the Runge-Kutta method.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)
All of the reactions were considered to be bimolecular and reversible, with kinetic parameters (association and dissociation rates) corresponding to those described below and listed in Tables II and III (as indicated in the footnotes). The initial concentrations of the reagents in the simulations were set at the steady state values corresponding to the contents of the two stopped-flow syringes immediately after mixing (time 0). The model assumes that the experimentally observable fluorescence change is associated with the species cTnCF27W N-domain·Ca2+, whereas all of the other species were considered fluorescently silent. EGTA koff(Ca) was experimentally determined to be 0.685 ± 0.002 s–1 (in the buffer utilized for the transient occupancy experiments) using Quin-2 at 15 °C. EGTA Kd(Ca) was fixed at 420 nM (31), with its kon(Ca) calculated to be 1.6317 x 106 M–1 s–1, using the equation kon(Ca) = koff(Ca)/Kd(Ca). EGTA Kd(Mg) was fixed at 39 mM, as determined by the program WEBMAXCLITE, version 1.15, under the buffer conditions utilized for the transient occupancy experiments (www.stanford.edu/~cpatton/maxc.html; Ref. 33). EGTA koff(Mg) was assumed to be 3000 s–1 with a kon(Mg) of 7.6923 x 104 M–1 s–1. Even though we could not experimentally determine EGTA koff(Mg) or kon(Mg), simulations indicated that by fixing the Kd(Mg) at 39 mM, the two kinetic parameters could be changed over a 100-fold range with no effect on the transient Ca2+ occupancy of each cTnCF27W protein. For the cTnCF27W mutants from which koff(Mg) was too rapid to be measured, it was assumed to be 3000 s–1. Simulations using the experimentally determined Kd(Mg) for each cTnCF27W protein indicated that koff(Mg) could be increased 10-fold or decreased to a measurable rate (≤1500 s–1) with negligible effects on the transient occupancy (after the mixing was complete). All of the other steady state and kinetic parameters were experimentally determined as described under "Results" and as summarized in Tables II and III. Each ACT was independently simulated by fluctuating only the kon(Ca) for each cTnCF27W protein, until the maximal and minimal values of kon(Ca) were found that matched the standard deviations from the mean transient occupancy, experimentally determined as described above. Each simulated kon(Ca) from the different ACTs was then used to calculate the average kon(Ca) for the N-domains of the cTnCF27W proteins.


View this table:
[in this window]
[in a new window]
  		TABLE II
Summary of Ca2+ binding properties for cTnCF27W and its mutants in the presence of 3 mM Mg2+

 


View this table:
[in this window]
[in a new window]
  		TABLE III
Summary of Mg2+ binding properties for cTnCF27W and its mutants

 
Calculation of Solvent Accessibilities—The protein analysis software MOLMOL (34) was used to calculate the percentage of solvent-accessible surface area (SA) ± S.D. for each of the five selected hydrophobic residues in the N-domain fragment of human cTnC (Table I). The average SA for each native residue was tabulated from the 40 apo or 40 Ca2+-bound NMR structures of the N-domain fragment (residues 1–89) of human cTnC available from the Protein Data Bank (1SPY [PDB] and 1AP4 [PDB] (15)). The percentage of SA for each native residue was calculated as the SA of the residue in the context of the protein structure divided by the SA of this residue extracted from the protein structure, with the resulting quotient multiplied by 100.


View this table:
[in this window]
[in a new window]
  		TABLE I
Summary of Ca2+ binding properties for cTnCF27W and its mutants in the absence of Mg2+

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Measurement of the Ca2+ Binding Affinities for cTnCF27W and Its Mutants in the Absence and Presence of 3 mM Mg2+— cTnCF27W undergoes an 1.44 ± 0.04-fold increase in Trp fluorescence (at 345 nm) upon Ca2+ binding to its N-terminal regulatory site (data not shown). To design cTnCF27W mutants with increased Ca2+ sensitivity of the regulatory domain, we individually substituted Phe20, Val44, Met45, Leu48, and Met81 (selected based on their location within the tertiary structure of the protein) with polar Gln (Fig. 1). The Ca2+-induced increases in Trp fluorescence, which occur when Ca2+ binds to the regulatory domain of cTnCF27W, F20QcTnCF27W, V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W, are shown in Fig. 2A (absence of Mg2+, summary in Table I) and Fig. 2B (presence of 3 mM Mg2+, summary in Table II). In the absence of Mg2+ (Fig. 2A), cTnCF27W exhibited a half-maximal Ca2+-dependent increase in its Trp fluorescence at 7 ± 1 µM. In the absence of Mg2+, at lower [Ca2+]free, the titration curves for M45QcTnCF27W and M81QcTncF27W could not be adequately fit by a single Hill equation, most likely because of the fact that Trp27 in these mutants also reported Ca2+ binding to their C-domain sites. The Ca2+ affinities for the regulatory N-domains of the mutant proteins ranged from 3.4 ± 0.3 µM for F20Q TnCF27W to 0.46 ± 0.04 µM for V44QcTnCF27W. Thus, introduction of polar Gln at position 20, 44, 45, 48, or 81 produced ~2.1–15.2-fold increases in the Ca2+ affinity of the regulatory site of cTnCF27W. In the presence of 3 mM Mg2+ (Fig. 2B), cTnCF27W exhibited a half-maximal Ca2+-dependent increase in its Trp fluorescence at 24 ± 1 µM. Thus, 3 mM Mg2+ produced 3.4-fold decrease in the Ca2+ sensitivity of the regulatory domain of cTnCF27W. Assuming competitive Mg2+ binding, the Kd(Mg) of the regulatory domain of cTnCF27W was calculated to be 1.2 ± 0.2 mM. In the presence of 3 mM Mg2+, the Ca2+ affinities for the mutants ranged from 9.5 ± 0.9 µM for F20QcTnCF27W to 1.62 ± 0.06 µM for V44QcTnCF27W. Thus, similar to the results in the absence of Mg2+, introduction of polar Gln at position 20, 44, 45, 48, or 81 produced ~2.5–14.8-fold increases in the Ca2+ affinity of the regulatory domain of cTnCF27W, in the presence of 3 mM Mg2+. Again, assuming competitive Mg2+ binding, the Kd(Mg) for the mutants ranged from 1.2 ± 0.1 mM for V44QcTnCF27W to 2.7 ± 0.3 mM for M45QcTnCF27W (summary in Table III). Thus, selected hydrophobic residue substitutions in the regulatory domain of cTnCF27W dramatically increase Ca2+ but not Mg2+ affinity. The Hill coefficients for all the Ca2+ titrations (in the absence or presence of 3 mM Mg2+) were between 0.76 and 1.3, consistent with binding of a single Ca2+ ion to the N-domain of cTnCF27W and its mutants (summary in Tables I and II).



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1.
Schematic representation of the regulatory domain of cTnCF27W. The cartoon depicts the amino acids in the regulatory N-domain (residues 1–89) of cTnCF27W that form the defunct site I, the functional Ca2+-binding site II, and the various helices (N–D). The black circles represent the hydrophobic residues that were individually mutated to Gln, excluding Trp27. All of the experimental data were obtained using full-length cTnCF27W, but the C-domain is not shown for the sake of simplicity.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2.
Ca2+ and Mg2+ binding to cTnCF27W and its mutants. A shows the Ca2+ dependent increases in Trp fluorescence for cTnCF27W ({square}), F20QcTnCF27W (), V44QcTnCF27W ({triangleup}), M45QcTnCF27W (*), L48QcTnCF27W ({blacksquare}), and M81QcTnCF27W ({circ}) as a function of –Log[Ca2+]. Microliter amounts of Ca2+ were added to 1 ml of each protein (0.6 µM) in 200 mM MOPS, 90 mM KCl, 2 mM EGTA, 1 mM DTT, pH 7.0, at 15 °C. Trp fluorescence emission was monitored at 345 nm with excitation at 285 nm. 0% Trp fluorescence corresponds to the apo state fluorescence, whereas 100% Trp fluorescence corresponds to the highest fluorescent state in the presence of Ca2+ for each individual cTnCF27W protein. Each data point represents a mean ± S.D. of three to five titrations fit with a logistic sigmoid. B shows the results of experiments identical to those in A, except the buffer contained 3 mM MgCl2. C shows the Mg2+-dependent decrease in Trp fluorescence of cTnCF27W({square}) at pCa 5.0, F20QcTnCF27W () at pCa 5.1, V44QcTnCF27W ({triangleup}) at pCa 5.4, M45QcTnCF27W (*) at pCa 5.3, L48QcTnCF27W ({blacksquare}) at pCa 5.4, and M81QcTnCF27W ({circ}) at pCa 5.3 as a function of –Log[Mg2+]. Initially each protein (0.6 µM) was subjected to a specified amount of [Ca2+]free, then titrated with increasing [Mg2+] in the same buffer as in A. Trp fluorescence emission was monitored at 345 nm with excitation at 285 nm. 100% Trp fluorescence corresponds to the Ca2+-bound state at the specified pCa, whereas 0% Trp fluorescence corresponds to the Mg2+-saturated state in the presence of the specified pCa.

 
Second Method for Measuring the Mg2+ Binding Affinities for the cTnCF27W and Mutants—Fig. 2C shows that addition of Mg2+ to cTnCF27W and its mutants (greater than 60% saturated with Ca2+) reversed the Ca2+-induced increase in Trp fluorescence in a concentration-dependent matter. Knowing Kd(Ca) and assuming competitive Mg2+ binding, the Kd(Mg) for cTnCF27W was calculated to be 1.9 ± 0.4 mM. The calculated Mg2+ affinities for the mutants ranged from 2.4 ± 0.7 mM for V44QcTnCF27W to 3.5 ± 0.5 mM for M45QcTnCF27W (summary in Table III). These values are in excellent agreement with the Mg2+ affinities calculated from the shifts in Ca2+ sensitivities, as described in the previous paragraph. Thus, using two different methods, the selected hydrophobic residue substitutions had only marginal effect on the Mg2+ affinity of the regulatory site of cTnCF27W.

Measurements of Ca2+ Dissociation Rates Using Trp and Quin-2 Fluorescence in the Absence and Presence of 3 mM Mg2+—Fluorescence stopped-flow measurements were conducted to determine the rate of Ca2+ dissociation from the regulatory domain of TnCF27W and its mutants. Fig. 3A shows the time course of the EGTA-induced decreases in Trp fluorescence as Ca2+ was removed from cTnCF27W, F20QcTnCF27W, V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W in the presence of 3 mM Mg2+. At 15 °C, excess EGTA removed Ca2+ from cTnCF27W at 1263 ± 188 s–1. For the mutants, the Ca2+ dissociation rates ranged from 1118 ± 113 s–1 for F20QcTnCF27W to 473 ± 14 s–1 for V44QcTnCF27W (summary in Table II). Thus, substitution of hydrophobic residues in position 20, 44, 45, 48, or 81 with polar Gln produced only ~1.1–2.7-fold decreases in the rate of Ca2+ dissociation from the regulatory site of cTnCF27W. Similar Ca2+ dissociation rates were measured in the absence of Mg2+ (data not shown; summary in Table I). These results were consistent with the Ca2+ dissociation rate from the regulatory domain of sTnCF29W not being affected by Mg2+ (8), again indicative of competitive binding of Mg2+ to the regulatory site of cTnCF27W.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 3.
Rates of Ca2+ dissociation from cTnCF27W and its mutants. A shows the time course of the decrease in Trp fluorescence as Ca2+ was removed by EGTA from cTnCF27W, F20QcTnCF27W, V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W at 15 °C. Each protein (2 µM) with 500 µM Ca2+ in 10 mM MOPS, 90 mM KCl, 3 mM MgCl2, and 1 mM DTT, pH 7.0, was rapidly mixed with an equal volume of EGTA (10 mM) in the same buffer. Trp fluorescence was monitored through a UV-transmitting black glass filter (UG1 from Oriel, Stratford, CT) with excitation at 285 nm. All of the kinetic traces were triggered at time 0, and the first ~1. 6 ms of premixing is shown. The traces were fit with a single exponential after mixing was complete (variance < 8 x 10–4). The traces have been normalized and displaced vertically for clarity. B shows the time course of the increase in Quin-2 fluorescence as Ca2+ was removed by Quin-2 from the N-terminal site of cTnCF27W, F20QcTnCF27W, V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W at 15 °C. Each protein (8 µM) with 45 µM Ca2+ in the same buffer as in A was rapidly mixed with an equal volume of Quin-2 (150 µM) in the same buffer. Quin-2 fluorescence was monitored through a 510-nm broad band pass interference filter with excitation at 330 nm. The traces were fit with a single exponential after mixing was complete (variance < 9 x 10–5). The traces are not normalized but have been displaced vertically for clarity. C shows the time course of the decrease in Trp fluorescence as Ca2+ was removed by EGTA from cTnCF27W and F20QcTnCF27W at 4 °C (Trp traces). Each protein (2 µM) with 500 µM Ca2+ in the same buffer as in A was rapidly mixed with an equal volume of EGTA (10 mM) in the same buffer. The traces have been normalized and displaced vertically for clarity. C also shows the time course of the increase in Quin-2 fluorescence as Ca2+ was removed by Quin-2 from the N-terminal site of cTnCF27W and F20QcTnCF27Wat 4 °C (Quin-2 traces). Each protein (8 µM) with 45 µM Ca2+ in the same buffer as in A was rapidly mixed with an equal volume of Quin-2 (150 µM) in the same buffer. The traces have been normalized and displaced vertically for clarity.

 
To verify that the Trp signal changes were accurately reporting the true Ca2+ dissociation rates and not a slower or faster structural change, Ca2+ dissociation rates were also measured using the fluorescent Ca2+ chelator Quin-2. Quin-2 fluorescence reported Ca2+ dissociation from both the N- and C-domains of cTnCF27W. However, the Ca2+ dissociation rates from the N-domain site of cTnCF27W and its mutants were easily distinguished from the Ca2+ dissociation rates from the C-domain sites (on average 0.772 ± 0.007s–1) because the latter rates were >570-fold slower. Fig. 3B shows the time course of the increases in Quin-2 fluorescence as Ca2+ dissociated from the N-domain site of cTnCF27W, F20QcTnCF27W, V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W at >1000 s–1, >1000 s–1, 444 ± 28 s–1, 691 ± 64 s–1, 476 ± 22 s–1, and 818 ± 77 s–1, respectively (in the presence of 3 mM Mg2+; summary in Table II). Fitting the traces with a double exponential over longer times (to account for the Ca2+ dissociation from the C-domain sites) gave similar N-domain rates (data not shown). Rates of Ca2+ dissociation from cTnCF27W and F20QcTnCF27W were too fast to reliably measure using Quin-2 fluorescence at 15 °C, so they were measured at 4 °C (Fig. 3C). At 4 °C, Quin-2 reported the rate of Ca2+ dissociation from the N-domain of cTnCF27W at 1015 ± 297 s–1. This rate is in good agreement with the previously reported rate of Ca2+ dissociation from the unlabeled chicken cTnC (772 ± 10 s–1), which was also obtained following the fluorescence of Quin-2 at 4 °C (35). In addition, the rates of Ca2+ dissociation from cTnCF27W and F20QcTnCF27W measured using Quin-2 fluorescence were nearly identical to the rates measured following the EGTA-induced Trp changes at 4 °C (Fig. 3C). The rates of Ca2+ dissociation from V44QcTnCF27W, M45QcTnCF27W, L48QcTnCF27W, and M81QcTnCF27W measured using Quin-2 fluorescence were also nearly identical to the rates following EGTA-induced Trp changes at 4 °C (data not shown; summary in Table II). Therefore, Trp27 accurately reported the rate of Ca2+ dissociation from the regulatory site of TnCF27W and its mutants. Measuring the rates of EGTA-induced Ca2+ dissociation at 15 and 4 °C enabled us to calculate the temperature coefficient Q10 for cTnCF27W and its mutants (summary in Table I). cTnCF27Wexhibited a Q10 of 1.2 ± 0.2, whereas for the rest of the mutants, Q10 ranged from 1.3 ± 0.1 for F20QcTnCF27W to 3.3 ± 0.2 for V44QcTnCF27W.

Calculations of Ca2+ Association Rates to the N-domain Site of TnCF27W and Its Mutants—Knowing the affinity of Ca2+ (Kd(Ca)) from the Ca2+ dependence of the increase in Trp27 fluorescence, and the rate of Ca2+ dissociation (koff(Ca)), we could calculate the rate of Ca2+ association (kon(Ca)) using the simple equation kon(Ca) = koff(Ca)/Kd(Ca). The calculated value of kon(Ca) for cTnCF27W was 1.7 ± 0.3 x 108 M–1 s–1. The calculated values of kon(Ca) for the mutants ranged from 2.8 ± 0.3 x 108 for F20QcTnCF27W to 8.8 ± 1.4 x 108 M–1 s–1 for V44QcTnCF27W (summary in Table I). Thus, substitution of hydrophobic residues in positions 20, 44, 45, 48, and 81 with polar Gln led to ~1.6–5.2-fold increases in calculated kon(Ca).

Using ACTs to Estimate the Ca2+ Association Rates to the N-domain Site of cTnCF27W in the Presence of 3 or 12 mM Mg2+—Because koff(Ca) is too fast to allow the experimental measurements of kon(Ca) using the standard pseudo-first order method in a stopped-flow apparatus (6), we estimated kon(Ca) by subjecting cTnCF27W and its mutants to ACTs of increasing duration and amplitude (8). The measurements were conducted in the presence of 3 or 12 mM Mg2+, to ensure complete Mg2+ occupation of the C-domain sites. We were unable to adequately simulate the data obtained in the absence of Mg2+, possibly because of the interference of the C-domain sites through interdomain interactions (data not shown). However, because Mg2+ binding to the regulatory domain of cTnCF27W or its mutants is competitive, the simulation of the experimental data obtained in the presence of Mg2+ estimates the true Ca2+ association rate, independent of [Mg2+]. Fig. 4A shows the time course of Ca2+ binding and subsequent dissociation from the N-domain site of cTnCF27W subjected to ACTs of increasing duration and amplitude at 15 °C in the presence of 3 mM Mg2+. When 10 µM Ca2+ was mixed with an equal volume of 2 µM TnCF27W in the presence of 1 mM EGTA, Ca2+ bound and transiently occupied 5 ± 1% (at 2 ms) of the regulatory site within cTnCF27W. Because Ca2+ was subsequently chelated by EGTA and removed from cTnCF27W, the fluorescence decayed back to its Ca2+-free fluorescence level. As the [Ca2+] was successively increased to 50, 100, 250, 500, 750, and 5000 µM (Fig. 4A), Ca2+ transiently occupied 17 ± 1 (at 2 ms), 29 ± 3 (at 2 ms), 44 ± 4 (at 2.8 ms), 70 ± 3 (at 2.5 ms), 81 ± 2 (at 2.5 ms), and 100% of the N-terminal regulatory site of TnCF27W, respectively. Because the percentage of occupancy of Ca2+ bound to the N-domain site of cTnCF27W during an ACT of given duration and amplitude is directly related to the Ca2+ association rate to this site, we were able to estimate the kon(Ca) to the regulatory site of cTnCF27W. Each individual trace was simulated using the computer program KSIM, which fixed the Ca2+ dissociation rate from cTnCF27W at 1263 s–1 and let the Ca2+ association rate vary until the modeled transient occupancy approximated the experimental data, taking into account the experimental error. The kinetic parameters used in the simulations are summarized under "Experimental Procedures" (Tables II and III). The final kon(Ca) was calculated as a mean of kon(Ca) for each individual trace ± S.D. The modeling predicted that we should observe the percentage of occupancy obtained experimentally if the kon(Ca) to the N-domain site of cTnCF27W was 1.2 ± 0.3 x 108 M–1 s–1. This value is in excellent agreement with the calculated value of 1.7 ± 0.3 x 108 M–1 s–1. The simulation using kon(Ca) of 1. 2 x 108 M–1 s–1 (Fig. 4A, {square}) is shown overlaying the experimental data. Fig. 4B demonstrates that the data is extremely sensitive to the value of kon(Ca). For instance, when 500 µM Ca2+ was mixed with an equal volume of 2 µM cTnCF27W in the presence of 1 mM EGTA, Ca2+ bound and transiently occupied 70 ± 3% (at 2.5 ms) of the regulatory site of cTnCF27W. Fig. 4B also shows computer simulations of the transient occupancy of cTnCF27W under the same conditions but assuming 2-fold slower ({blacktriangleup}) and 2-fold faster ({blacktriangledown}) kon(Ca). Modeling with 0.6 x 108({blacktriangleup}), 1.2 x 108 ({square}), and 2.4 x 108 M–1 s–1 ({blacktriangledown}) kon(Ca) suggests that 53, 68, and 81% of the N-domain site would be occupied by Ca2+ after mixing was complete at 2.5 ms. Clearly, the simulation demonstrates how small changes in the Ca2+ association rate can produce large changes in the percentage of occupancy of the Ca2+-binding site.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4.
Percentage of Ca2+ occupancy of the N-terminal site of cTnCF27W during artificial Ca2+ transients of different amplitude and duration. A shows the time courses of the decrease in Trp fluorescence when cTnCF27W (2 µM) in the presence of 1 mM EGTA in 10 mM MOPS, 90 mM KCl, 3 mM MgCl2, 1 mM DTT, pH 7.0, at 15 °C was rapidly mixed with increasing Ca2+ ranging from 10 to 5000 µM in the same buffer. Each trace represents an average of three separate experiments, each averaging at least 10 separate traces. The specific value of [Ca2+] before mixing for each ACT is shown under the trace. The cTnCF27W transient occupancy was simulated using the program KSIM (32) and is shown ({square}) overlaying the experimental data. The initial [EGTA] was set to 500 µM, and the initial [Ca2+] was set to values ranging from 5 to 2500 µM, to mimic the experimental data after mixing. B shows the time course of the decrease in Trp fluorescence when cTnCF27W (2 µM) in the presence of 1 mM EGTA in the same buffer as in A was rapidly mixed with 500 µM Ca2+ in the same buffer at 15 °C. Computer simulations of the cTnCF27W transient occupancy ({square}) are shown overlaying the experimental data. The initial [EGTA] was set at 500 µM, and the initial [Ca2+] was set to 250 µM to mimic the experimental data after mixing. B also shows the computer simulations of the transient occupancy for the protein with 2-fold slower ({blacktriangleup}) or 2-fold faster ({blacktriangledown}) kon(Ca), compared with cTnCF27W. C shows the results for the experiments identical to those in A, except the buffer contained 12 mM MgCl2.

 
To examine the effect of higher [Mg2+] on the transient occupancy of cTnCF27W by Ca2+, cTnCF27W was subjected to ACTs of increasing duration and amplitude in the presence of 12 mM Mg2+. Fig. 4C shows the time course of Ca2+ binding and subsequent dissociation from the N-domain site of cTnCF27W subjected to ACTs of increasing duration and amplitude at 15 °C in the presence of 12 mM Mg2+. As the [Ca2+] was successively increased from 10 to 50, 100, 250, 500, 750, and 5000 µM (Fig. 4C), Ca2+ transiently occupied 4 ± 3 (at 2 ms), 9 ± 4 (at 2 ms), 16 ± 4 (at 2 ms), 25 ± 3 (at 2.8 ms), 48 ± 4 (at 2.5 ms), 62 ± 2 (at 2.5 ms), and 100% of the N-terminal regulatory site of cTnCF27W, respectively. Thus, higher [Mg2+] leads to a decreased percent occupancy of the regulatory site of cTnCF27W, because of a larger percentage of the N-domain of cTnCF27W being initially occupied by Mg2+. The simulation using kon(Ca) of 1.2 x 108 M–1 s–1 in the presence of 12 mM Mg2+ ({square}) is shown overlaying the experimental data. Therefore, the kon(Ca) estimated from the experimental data in the presence of 3 mM Mg2+ could be used to accurately predict the transient occupancy of cTnCF27W in the presence of 12 mM Mg2+. Furthermore, these data support the conclusion that Mg2+ competitively binds to the N-domain of cTnCF27W with 1.2 mM affinity.

Using ACTs to Estimate the Ca2+ Association Rates to the N-domain Site of cTnCF27W Mutants in the Presence of 3 mM Mg2+—Fig. 5A shows the time course of Ca2+ binding and subsequent dissociation from the N-domain site of V44QcTnCF27W subjected to ACTs of increasing duration and amplitude at 15 °C in the presence of 3 mM Mg2+. As the [Ca2+] was successively increased from 5 to 10, 25, 100, 250, 500, 750, and 2000 µM (Fig. 4A), Ca2+ transiently occupied 28 ± 3 (at 2 ms), 38 ± 3 (at 3 ms), 58 ± 3 (at 3 ms), 78 ± 4 (at 3.5 ms), 85 ± 4 (at 4.3 ms), 86 ± 3 (at 7.5 ms), 92 ± 2 (at 10 ms), and 100% of the N-terminal regulatory site of V44QcTnCF27W, respectively. Each individual trace was simulated using the computer program KSIM, which fixed the Ca2+ dissociation rate from V44QcTnCF27W at 473 s–1 and let the Ca2+ association rate vary until the modeled transient occupancy approximated the experimental data. The simulation predicted that we should observe the percentage of occupancy obtained experimentally if the kon(Ca) to the N-domain site of V44QcTnCF27W was 9.6 ± 1 x 108 M–1 s–1. The simulation using kon(Ca) of 9.6 x 108 M–1 s–1 ({triangleup}) is shown overlaying the experimental data. Clearly, the Ca2+ association rate to the N-domain of cTnCF27W can be increased and thus is not diffusion-limited, as previously suggested for sTnCF29W (6).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 5.
Percent Ca2+ occupancy of the N-terminal site of cTnCF27W mutants during artificial Ca2+ transients of different amplitude and duration. A shows the time courses of the decrease in Trp fluorescence when V44QcTnCF27W (2 µM) in the presence of 1 mM EGTA in 10 mM MOPS, 90 mM KCl, 3 mM MgCl2, 1 mM DTT, pH 7.0, at 15 °C was rapidly mixed with increasing Ca2+ ranging from 5 to 2000 µM in the same buffer. Each trace represents an average of three separate experiments, each averaging at least 10 separate traces. The specific [Ca2+] before mixing for each ACT is shown under the trace. Computer simulations of the V44QcTnCF27W transient occupancy ({triangleup}) are shown overlaying the experimental data. The initial [EGTA] was set to 500 µM, and the initial [Ca2+] was set to values ranging from 2.5 to 1000 µM, to mimic the experimental data. B shows the time courses of the decrease in Trp fluorescence when M81QcTnCF27W (2 µM) in the presence of 1 mM EGTA in the same buffer as in A was rapidly mixed with increasing Ca2+ ranging from 5 to 2000 µM in the same buffer. Each trace represents an average of three separate experiments, each averaging at least 10 separate traces. The specific [Ca2+] immediately after mixing for each ACT is shown under the trace. Computer simulations of M81QcTnCF27W transient occupancy ({circ}) are shown overlaying the experimental data. The initial [EGTA] was set to 500 µM, and the initial [Ca2+] was set to values ranging from 2.5 to 1000 µM to mimic the experimental data after mixing. C shows the time courses of the decreases in Trp fluorescence when cTnCF27W, V44QcTnCF27W, and M81QcTnCF27W in the presence of 1 mM EGTA in the same buffer as in A were rapidly mixed with 50 µM Ca2+. Computer simulations for cTnCF27W ({square}), V44QcTnCF27W ({triangleup}), and M81QcTnCF27W ({circ}) are shown overlaying the experimental data. The initial [EGTA] was set to 500 µM, and the initial [Ca2+] was set to 25 µM to mimic the experimental data after mixing.

 
Fig. 5B shows the time course of Ca2+ binding and subsequent dissociation from the N-domain site of M81QcTnCF27W subjected to ACTs of increasing duration and amplitude at 15 °C in the presence of 3 mM Mg2+. As the [Ca2+] was successively increased from 5 to 10, 25, 100, 250, 500, 750, and 2000 µM (Fig. 4A), Ca2+ transiently occupied 10 ± 2.5 (at 2.5 ms), 19 ± 3 (at 2 ms), 32 ± 4 (at 2.5 ms), 59 ± 5 (at 3 ms), 73 ± 4 (at 3.5 ms), 82 ± 6 (at 5 ms), 78 ± 4 (at 10 ms), and 100% of the N-terminal regulatory site of M81QcTnCF27W, respectively. Each individual trace was simulated using the computer program KSIM, which fixed the Ca2+ dissociation rate from M81QcTnCF27W at 1105 s–1 and let the Ca2+ association rate vary until the modeled transient occupancy approximated the experimental data. The simulation predicted that we should observe the percentage of occupancy obtained experimentally if the kon(Ca) to the N-domain site of M81QcTnCF27W was 5.3 ± 0.5 x 108 M–1 s–1. The simulation using kon(Ca) of 5.3 x 108 M–1 s–1 ({circ}) is shown overlaying the experimental data.

Fig. 5C compares the time course of transient Ca2+ binding to the N-domain site of cTnCF27W, V44QcTnCF27W, and M81QcTnCF27W subjected to identical ACTs at 15 °C in the presence of 3 mM Mg2+. When 50 µM Ca2+ was mixed with an equal volume of each protein (2 µM) in the presence of 1 mM EGTA, Ca2+ bound and transiently occupied 17 ± 1 (at 2 ms), 67 ± 3 (at 3.5 ms), and 49 ± 3% (at 2.5 ms) of the regulatory site of cTnCF27W, V44QcTnCF27W, and M81QcTnCF27W, respectively. The computer simulations for cTnCF27W ({square}), V44QcTnCF27W ({triangleup}), and M81QcTnCF27W ({circ}) are shown overlaying the experimental data. Clearly, Ca2+-sensitizing mutants exhibit a dramatically increased percentage of occupancy of their regulatory site, relative to cTnCF27W, when subjected to identical ACTs, because of their drastically faster Ca2+ association rates. Table I summarizes the kon(Ca) for the rest of the mutants estimated using ACTs. The simulated kon(Ca) ranged from 3.1 ± 0.5 x 108 for F20QcTnCF27W to 10.4 ± 0.8 x 108 M–1 s–1 for L48QcTnCF27W (summary in Table I). These values are in excellent agreement with the values of kon(Ca) calculated using the equation kon(Ca) = koff(Ca)/Kd(Ca) (Table I). Thus, substitution of hydrophobic residues in positions 20, 44, 45, 48, or 81 with polar Gln produced ~2.6–8.7-fold increases in kon(Ca).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
EF-hand Ca2+-binding proteins exhibit a wide range of Ca2+ binding affinities (>106-fold variation) and dissociation rates (>104-fold variation) (for review, see Refs. 36 and 37). The mechanisms controlling the vast range of EF-hand Ca2+ binding affinities and exchange rates remain elusive. Clearly, helical and Ca2+ chelating and nonchelating loop residues can have a dramatic influence on cation affinity and exchange rates (8, 24, 25, 38–41). The goal of this study was to design cTnC mutants with increased Ca2+ affinity by substitution of selected hydrophobic residues in specific locations within the tertiary structure of the protein with polar Gln. To follow Ca2+ binding and exchange with the regulatory domain of cTnC, we utilized the intrinsic fluorescence of cTnCF27W, which has been previously used to study Ca2+ binding to the N-domain site of cTnC and its mutants or isoforms (26–29). This mutation is analogous to sTnCF29W, which enabled numerous studies of Ca2+ and target protein binding to the N-domain of sTnC and its mutants (6, 8, 22, 25, 42).

Previously, we examined the role of hydrophobic residues in Ca2+ binding and exchange with the regulatory domain of sTnCF29W by individually substituting 27 hydrophobic Phe, Ile, Leu, Val, and Met residues with polar Gln (25). The results demonstrated that individual substitution of hydrophobic residues with polar Gln could both dramatically increase or decrease the Ca2+ affinity of the regulatory domain of sTnCF29W, depending on the location of the residue within the tertiary structure of the protein (25). No correlation was found between the Ca2+ affinity of the 27 mutants and the solvent accessibility of the mutated residues in either the absence or presence of Ca2+ (or the difference between the two states), indicating that the changes in solvent exposure were not the sole determinant in how the residue affects Ca2+ binding. Substitution of Phe22, Val45 Met46, Leu49, or Met82 with polar Gln led to large (3.2–18.9-fold) increases in the Ca2+ affinity. We concluded that these mutations increased Ca2+ affinity because they shift the equilibrium from the apo state toward the Ca2+-bound state by reducing the hydrophobic contact between the BC and NAD units in the apo state. However, the N-domain of cTnC remains essentially closed in the Ca2+ bound state, because the BC unit moves away only slightly from the NAD unit (15). Therefore, analogous residues in cTnC, Phe20, Val44, Met45, Leu48, and Met81 exhibit no increase in their solvent accessibility upon Ca2+ binding (Table I) and form numerous side chain contacts with each other in both the apo and Ca2+-bound states. Thus, there was a question of whether analogous mutations in cTnC would have the same effect on Ca2+ affinity and exchange rates as they did in sTnC.

The half-maximal Ca2+ binding to cTnCF27W occurred at 7 µM at 15 °C. This value is in a range of previously reported Ca2+ affinities for bovine cTnCF27W at 7 °C (10.7 µM) and 21 °C (5.1 µM) (27). Residues Phe20 and Met81 are located within the NAD unit, whereas Val44, Met45, and Leu48 are located within the BC unit. All of these residues are almost completely buried in the absence and presence of Ca2+ (with an exception of Leu48) (Table I and Ref. 15), with their side chains involved in extensive hydrophobic interactions between the BC and NAD units (Fig. 6). However, upon TnI147–163 binding to the regulatory domain of cTnC in the presence of Ca2+, all of the interunit hydrophobic interactions are lost as the BC unit moves away from the NAD unit (Fig. 6 and Ref. 17). Thus, substitution of either one of these residues with polar Gln could increase Ca2+ affinity by facilitating the movement of the BC unit away from the NAD unit, mimicking the effects of cTnI binding. Indeed, the mutants exhibited ~2.1–15.2-fold increases in their Ca2+ affinity, ranging from 3.4 µM for F20QcTnCF27W to 0.46 µM for V44QcTnCF27W. In this respect, the regulatory domain of cTnC behaved similarly to the regulatory domains of both sTnC and CaM, in which decreasing the hydrophobic interactions between the BC and (N)AD units in the apo state, through similar mutations, led to increased Ca2+ affinity (22, 23, 25, 43). Complementary to these findings, stabilizing the interactions between the BC and (N)AD units of sTnC or CaM, either through disulfide bond, salt bridge formation, or substitution of buried polar residues with hydrophobic Leu or Ile, led to decreased Ca2+ affinity (44–48). Our findings further support the idea that a change in solvent accessibility of a hydrophobic residue may not be the main determinant for how a mutation of this residue affects the Ca2+ binding properties of an EF-hand protein (25).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 6.
Location of Ca2+ sensitizing mutations in the regulatory domain of cTnC. The NMR structure of the apo N-domain fragment (residues 1–89) of human cTnC is shown on the left (Protein Data Bank 1SPY [PDB] , model 13 (15)). The NMR structure of the Ca2+-bound N-domain fragment (residues 1–89) of human cTnC is shown in the middle (Protein Data Bank file 1AP4 [PDB] , model 14 (15)). The NMR structure of the Ca2+ and TnI147–163 bound N-domain fragment (residues 1–89) of human cTnC is shown on the right (Protein Data Bank file 1MXL [PDB] , model 18 (17)). The cTnI147–163 is not shown for the sake of clarity. The models shown in this figure are those stated to be the best representatives of the ensemble conformer (15, 17). This figure was drawn using Rasmol (61). The helices are labeled (N, A, B, C, and D) and shown as ribbons; hydrophobic residues that were mutated to Gln (Phe20, Val44, Met45, Leu48, and Met81) are shown in a ball-and-stick format and labeled.

 
The five cTnCF27W mutants studied in the present work possessed ~2.1–15.2-fold higher Ca2+ affinities but only ~1.2–2.9-fold slower Ca2+ dissociation rates. In this respect, the cTnCF27W mutants behaved differently from hydrophobic core mutants of calbindin D9K, in which variations in the Ca2+ affinity were a consequence of the perturbations of the Ca2+ dissociation rate (24). Furthermore, Ca2+ sensitizing mutants in cTnCF27W behaved differently from analogous mutants of sTnCF29W and the intact CaM mutant with a Phe19 -> Trp mutation, in which the increase in Ca2+ affinity was largely due to a decrease in Ca2+ dissociation rate (25, 43). This unusual behavior of cTnC could perhaps be related to the fact that despite being a regulatory Ca2+ sensor, the N-domain of cTnC undergoes only a minor conformational transition upon Ca2+ binding. Calculation of the Ca2+ association rate using the equation kon(Ca) = koff(Ca)/Kd(Ca) suggested that the Ca2+-sensitizing mutations should cause ~1.7–5.3-fold increase in the Ca2+ association rates to the N-domain of cTnCF27W. Limitations of the stopped-flow technique would not allow us to directly measure the rate of Ca2+ association to the regulatory site of cTnCF27W using the standard pseudo-first order approach, because most of the reaction occurred during the dead time of the instrument. Previously, we demonstrated that by exposing Ca2+-binding proteins/enzymes to ACTs and examining the percent occupancy as a function of ACT duration and amplitude, we could experimentally determine the Ca2+ association rate (31). Thus, to verify that the Ca2+-sensitizing mutations increase the rate of Ca2+ association, cTnCF27W and its mutants were subjected to ACTs of increasing amplitude and duration. The experimental Ca2+ association rate to the regulatory domain of cTnCF27W was estimated at 1.2 x 108 M–1 s–1, in excellent agreement with the calculated Ca2+ association rate of 1.7 x 108 M–1 s–1. Our transient occupancy data were consistent with a one-step Ca2+ association process and were not consistent with either a two-step (35) or a three-step mechanism of Ca2+ association to the regulatory domain of cTnC (49). However, the intrinsic fluorescent Trp27 used in the present study is located on helix A, immediately preceding the first defunct Ca2+ binding loop, whereas Hazard et al. (35) and Dong et al. (49) utilized the extrinsic fluorescent probe IAANS linked to a single Cys84 on helix D of cTnCC35S. Thus, the different location and properties of the two probes could be a reason why Trp27 reports a true Ca2+ association rate and does not sense any slow conformational transitions. In fact, Hazard et al. (35) reported that the fluorescence of IAANS covalently linked to Cys84 of chicken cTnCC35S followed the rate of a slower conformational rearrangement rather than the faster true rate of Ca2+ dissociation. On the other hand, the fluorescence of Trp27 accurately reported the rate of Ca2+ dissociation from the regulatory site of cTnCF27W. However, the different location and properties of two fluorescent reporters did not affect the Ca2+-sensitizing effect of the five individual mutations studied in the present work, because these mutations also produced ~2.4–23-fold increases in Ca2+ binding to the N-domain of cTnCC35S labeled with IAANS on Cys84, which exhibited a Kd(Ca) of 7.5 µM (50).2

The experimentally measured and calculated Ca2+ association rates to the N-domain of cTnCF27W (~1.2–1.7 x 108 M–1 s–1, reported in this work) and sTnCF29W (~1.1–1.6 x 108 M–1 s–1; 8, 25) were nearly identical at 15 °C. The simulation of the transient occupancy data indicated that the Ca2+-sensitizing mutations increased the Ca2+ association rates to the N-domain of cTnCF27W ~2.6–8.7-fold. Clearly, the rate of Ca2+ association to the regulatory domain of cTnCF27W can be increased and thus is not diffusion-controlled as previously suggested for sTnCF29W (6). Faster Ca2+ association rates could indicate that the mutations decreased the energy of the transition state barrier (36), but this alone would not explain the altered equilibrium constants and lack of proportional changes in the Ca2+ dissociation rates. For the Ca2+-sensitizing mutations in cTnCF27W, there was a strong correlation between the Ca2+ association rate and Ca2+ affinity (r2 = 0.76), contrasting with the weak correlation (r2 = 0.26) observed for analogous mutations in sTnCF29W (Fig. 7A and Ref. 25). Thus, the mechanisms behind the enhanced Ca2+ sensitivity by analogous mutations in sTnCF29W and cTnCF27W were strikingly different. Clearly, Ca2+-sensitizing mutations in sTnCF29W primarily decreased Ca2+ dissociation, whereas analogous mutations in cTnCF27W drastically increased Ca2+ association. Because the changes in Ca2+ association rate potentially reflect the perturbation of the apo state, the data obtained in the present work are consistent with the idea that reducing the hydrophobic interactions between the BC and NAD units destabilized the apo state of the N-domain leading to the increased Ca2+ affinity. There is the possibility that these hydrophobic residue substitutions led to a slight opening of the N-domain in the apo state. On the other hand, the fact that Ca2+ dissociates slower from the Ca2+-sensitizing cTnCF27W mutants also suggests possible perturbations of the Ca2+-bound state. Interestingly, the Ca2+-sensitizing mutations in cTnCF27W, but not in sTnCF29W, increased the temperature coefficient Q10 for the rates of Ca2+ dissociation in the 4–15 °C range (Fig. 7B). It is possible that the Ca2+-sensitizing mutations studied in the present work caused the N-domain of cTnCF27W to be more open in the Ca2+-bound state, perhaps to some extent mimicking cTnI binding, which also produces an ~10-fold increase in the Ca2+ affinity of the regulatory site of cTnC (9). Clearly, structural studies are needed to examine whether the Ca2+-sensitizing mutations affect the tertiary structure of cTnC, as proposed in this study. Nuclear magnetic resonance studies are currently underway in collaboration with the laboratory of Dr. Paul Rosevear (University of Cincinnati).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7.
Comparison of kon(Ca) and Q10 for koff(Ca) between Ca2+ sensitizing cTnCF27W and sTnCF29W mutants. A shows the correlation between the Ca2+ association rate kon(Ca) and Ca2+ affinity Kd(Ca) for cTnCF27W (cTnCF27W, {square}), F20QcTnCF27W (F20Q, {square}), V44QcTnCF27W (V44Q, {square}), M45QcTnCF27W (M45Q, {square}), and M81QcTnCF27W (M81Q, {square}). The solid line was obtained by fitting the data with a linear regression. Each data point represents the mean ± S.D. of three to five separate experiments. The correlation coefficient (r2) was 0.76. A also shows the correlation between the Ca2+ association rate kon(Ca) and Ca2+ affinity Kd(Ca) for sTnCF29W (sTnCF29W, {blacksquare}), F22QsTnCF29W (F22Q, {blacksquare}), V45QsTnCF29W (V45Q, {blacksquare}), M46QsTnCF29W (M46Q, {blacksquare}), L49QsTnCF29W (L49Q, {blacksquare}), and M82QsTnCF29W (M82Q, {blacksquare}). The solid line was obtained by fitting the data with a linear regression. The correlation coefficient (r2) was 0.26. The data for sTnCF29W have been adapted from Tikunova et al. (25). These data are shown for comparative purposes, because it was collected under identical experimental conditions, except the Ca2+ association rate was calculated using the equation kon(Ca) = koff(Ca)/Kd(Ca). B shows the relationship between the temperature coefficient Q10 (for the Ca2+ dissociation rate in the 4–15 °C range; Q10 = (koff(Ca) at 15 °C/koff(Ca) at 4 °C)10/11) and Ca2+ affinity Kd(Ca) for cTnCF27W (cTnCF27W, {square}), F20Qc-TnCF27W (F20Q, {square}), V44QcTnCF27W (V44Q, {square}), M45QcTnCF27W (M45Q, {square}), and M81QcTnCF27W (M81Q, {square}). Each data point represents a mean ± S.D. of three to five separate experiments. B also shows the relationship between the temperature coefficient Q10 and Ca2+ affinity Kd for sTnCF29W (sTnCF29W, {blacksquare}), F22QsTnCF29W (F22Q, {blacksquare}), V45QsTnCF29W (V45Q, {blacksquare}), M46QsTnCF29W (M46Q, {blacksquare}), L49QsTnCF29W (L49Q, {blacksquare}), and M82QsTnCF29W (M82Q, {blacksquare}). The published data for sTnCF29W collected at 15 °C as previously described (25) and unpublished data collected under the same conditions (25) but at 4 °C were used to calculate Q10. Each data point represents a mean ± S.D. of three to five separate experiments. The dashed lines connecting the data points were used for visual reference.

 
The question of whether Mg2+ affects the Ca2+ sensitivity of actomyosin ATPase or force production in cardiac muscle is unresolved and controversial. Several studies have shown that increasing [Mg2+] in the millimolar range led to the decrease in Ca2+ sensitivity of myofibrillar actomyosin ATPase or force development in cardiac fibers (51–54). However, a recent study reported that increasing Mg2+ from 1 to 8 mM had no significant effect on the Ca2+ sensitivity of actomyosin ATPase activity and force production in skinned rat cardiac cells (55). The N-domain site of cTnC is generally considered to be Ca2+-specific under physiological Mg2+ concentrations. The question of whether Mg2+ binds to the N-domain site of cTnC and competes with Ca2+ is also unresolved. An early study reported that Mg2+ decreased the Ca2+ sensitivity of the regulatory site of fluorescently labeled cTnC, with a Kd(Mg) calculated to be ~3.8 mM at room temperature, assuming competitive Mg2+ binding (56). Another study, using equilibrium dialysis, concluded that Mg2+ did not compete with Ca2+ for the N-domain site of cTnC but suggested the presence of several auxiliary Mg2+-binding sites (9).

Unlike Ca2+, Mg2+ does not increase the fluorescence of cTnCF27W. However, under our experimental conditions, increasing Mg2+ from 0 to 3 mM reduced the Ca2+ sensitivity of the regulatory domain of cTnCF27W ~3.4-fold. In addition, Mg2+ was able to reverse the Ca2+-induced increase in fluorescence of cTnCF27W in a concentration-dependent manner. Furthermore, higher [Mg2+] led to a decrease in transient occupancy of cTnCF27W by Ca2+, suggesting increased occupation of the regulatory site by Mg2+. Thus, all of our results indicate that the regulatory site of cTnCF27W binds Mg2+ competitively with 1.2–1.9 mM affinity at 15 °C. Therefore, according to our data, the regulatory domain of cTnC would be 33–44% saturated at rest under physiological conditions of ~1mM Mg2+ (57) and 100 nm Ca2+, assuming that cTnC incorporated into cardiac muscle still possesses a similar ability to bind Mg2+. Interestingly, the Ca2+-sensitizing mutations had only marginal effects on the Mg2+ binding properties of the regulatory domain of cTnCF27W. These findings are not surprising, because Mg2+ binding to the EF-hands of Ca2+/Mg2+-binding proteins, such as calbindin D9K and CaM, affected only the local structural environment of the cation-binding loop (58–60). Thus, mutations of residues outside the loops are not expected to drastically modify Mg2+ affinity, unless they grossly perturb the structure of the loop itself.

Because cTnC does not regulate muscle mechanics in isolation but as a part of the cTn complex, it is important to elucidate the Ca2+/Mg2+-dependent interactions between cTnC and cTnI/cardiac troponin T. Studies aimed toward understanding whether mutations sensitizing isolated cTnC to Ca2+ also increase Ca2+ sensitivity of the cTn complex and muscle force development are currently underway in our laboratory. The main goal of our laboratory is to delineate the influence of the rates of Ca2+ exchange with the regulatory domain of TnC on the kinetics of striated muscle contraction and relaxation. Slowing of cardiac muscle relaxation would be an undesirable consequence of the increased sensitivity of contractile proteins to Ca2+. The cTnCF27W mutants designed for this study demonstrated that it is possible to drastically increase Ca2+ affinity of the regulatory domain without dramatically slowing the Ca2+ dissociation rate. These cTnC mutants could be used as molecular tools to examine whether it is possible to increase the Ca2+ sensitivity of force development with minimal effect on the rate of cardiac muscle relaxation. Perhaps, in the future, the cTnC mutant proteins with desired properties can be used for the treatment of heart disease to enhance cardiac contractility without further impairing relaxation.

In summary, we utilized the Phe27 -> Trp substitution to study Ca2+ binding and exchange with the regulatory site of cTnC. We then designed five Ca2+-sensitizing cTnCF27W mutants, in which we individually substituted hydrophobic Phe20, Val44, Met45, Leu48, or Met81 with polar Gln. None of the mutations affected the ability of cTnCF27W to competitively bind Mg2+ with a physiologically relevant affinity. The cTnCF27W mutants exhibited a ~2.1–15.2-fold increase in Ca2+ affinity, a ~1.2–2.9-fold decrease in Ca2+ dissociation rate, and a ~2.6–8.7-fold increase in Ca2+ association rate. The rational design of Ca2+-sensitizing cTnC mutants presented in this work is a first step toward understanding how the sequence of an EF-hand protein determines its Ca2+ binding properties, structure, and function.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants AR20792 (to Jack A. Rall) and HL073600 (to S. B. T.) and by an award from the American Heart Association, Ohio Valley Affiliate (to J. P. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, The Ohio State University, 209 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-4467; Fax: 614-292-4888; E-mail: tikunova1{at}hotmail.com.

1 The abbreviations used are: Tn, troponin; cTnC, intact human cardiac troponin C; sTnC, intact chicken skeletal troponin C; cTnCF27W, intact cTnC mutant with Phe27 -> Trp mutation; cTnCC35S, intact cTnC with Cys35 -> Ser mutation; sTnCF29W, intact sTnC mutant with Phe29 -> Trp mutation; TnI, troponin I; cTnI, cardiac troponin I; CaM, calmodulin; ACT, artificial Ca2+ transient; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; Quin-2, 2-((2-bis(carboxymethyl)amino-5-methylphenoxy)methyl)-6-methoxy-8-bis(carboxymethyl)aminoquionline; N-domain, N-terminal domain; C-domain, C-terminal domain; SA, surface area. Back

2 S. B. Tikunova and J. P. Davis, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jack A. Rall for support and critical reading of the manuscript, Dr. Lawrence Smillie for the generous gift of human cTnC plasmid, Dr. Peter Reiser for critical reading of the manuscript, Dr. Russ Hille for helpful discussion of the data, Dr. Wessel Dirksen for helping with Labworks 4.0 Software, and Craig McElroy for assistance with using the computer program MOLMOL.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gordon, A. M., Homsher, E., and Regnier, M. (2000) Physiol. Rev. 80, 853–924[Abstract/Free Full Text]
  2. Gomez, A. V., Potter, J. D., and Szczesna-Corday, D. (2002) IUBMB Life 54, 323–333[Medline] [Order article via Infotrieve]
  3. Nelson, M. R., and Chazin, W. J. (1998) Biometals 11, 297–318[CrossRef][Medline] [Order article via Infotrieve]
  4. Filatov, V. L., Katrukha, A. G., Bulgarina, T. V., and Gusev, N. B. (1999) Biochemistry (Mosc.) 64, 969–985[Medline] [Order article via Infotrieve]
  5. Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628–4633[Abstract/Free Full Text]
  6. Johnson, J. D., Nakkula, R. J., Vasulka, C., and Smillie, L. B. (1994) J. Biol. Chem. 269, 8919–8923[Abstract/Free Full Text]
  7. Ogawa, Y. (1985) J. Biochem. (Tokyo) 97, 1011–1023[Abstract/Free Full Text]
  8. Davis, J. P., Rall, J. A., Reiser, P. J., Smillie, L. B., and Tikunova, S. B. (2002) J. Biol. Chem. 277, 49716–49726[Abstract/Free Full Text]
  9. Holroyde, M. J., Robertson, S. P., Johnson, J. D., Solaro, R. J., and Potter, J. D. (1980) J. Biol. Chem. 255, 11688–11693[Abstract/Free Full Text]
  10. Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755–767[Abstract]
  11. Herzberg, O., and James, M. N. G. (1985) Nature 313, 653–659[CrossRef][Medline] [Order article via Infotrieve]
  12. Slupsky, C. M., and Sykes, B. D. (1995) Biochemistry 34, 15953–15964[CrossRef][Medline] [Order article via Infotrieve]
  13. Houdusse, A., Love, M. L., Dominguez, R., Grabarek, Z., and Cohen, C. (1997) Structure 5, 1695–1711[Medline] [Order article via Infotrieve]
  14. Sia, S. K., Li, M. X., Spyracopoulos, L., Gagne, S. M., Liu, W., Putkey, J. A., and Sykes, B. D. (1997) J. Biol. Chem. 272, 18216–18221[Abstract/Free Full Text]
  15. Spyracopoulos, L., Li, M. X., Sia, S. K., Gagne, S. M., Chandra, M., Solaro, R. J., and Sykes, B. D. (1997) Biochemistry 36, 12138–12146[CrossRef][Medline] [Order article via Infotrieve]
  16. Ikura, M. (1996) Trends Biochem. Sci. 21, 14–17[CrossRef][Medline] [Order article via Infotrieve]
  17. Li, M. X., Spyracopoulos, L., and Sykes, B. D. (1999) Biochemistry 38, 8289–8298[CrossRef][Medline] [Order article via Infotrieve]
  18. de Tombe, P. P. (1998) Cardiovasc. Res. 37, 367–380[Abstract/Free Full Text]
  19. Endoh, M. (1995) Gen. Pharmacol. 26, 1–31[Medline] [Order article via Infotrieve]
  20. Arteaga, G. M., Kobayashi, T., and Solaro, R. J. (2002) Ann. Med. 34, 248–258[CrossRef][Medline] [Order article via Infotrieve]
  21. Erhardt, L. R. (2003) Ital. Heart. J. 4, (Suppl. 2) 27S–33S.
  22. Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L. B. (1992) Biochemistry 31, 6545–6553[CrossRef][Medline] [Order article via Infotrieve]
  23. da Silva, A. C., de Araujo, A. H., Herzberg, O., Moult, J., Sorenson, M., and Reinach, F. C. (1993) Eur. J. Biochem. 213, 599–604[Medline] [Order article via Infotrieve]
  24. Kragelund, B. B., Jonsson, M., Bifulco, G., Chazin, W. J., Nilsson, H., Finn, B. E., and Linse, S. (1998) Biochemistry 37, 8926–8937[CrossRef][Medline] [Order article via Infotrieve]
  25. Tikunova, S. B., Rall, J. A., and Davis, J. P. (2002) Biochemistry 41, 6697–6705[CrossRef][Medline] [Order article via Infotrieve]
  26. Moyes, C. D., Borgford, T., LeBlanc, L., and Tibbits, G. F. (1996) Biochemistry 35, 11756–11762[CrossRef][Medline] [Order article via Infotrieve]
  27. Gillis, T. E., Marshall, C. R., Xue, X. H., Borgford, T. J., and Tibbits, G. F. (2000) Am. J. Physiol. 279, R1707–R1715
  28. Gillis, T. E., Blumenschein, T. M., Sykes, B. D., and Tibbits, G. F. (2003) Biochemistry 42, 6418–6426[CrossRef][Medline] [Order article via Infotrieve]
  29. Gillis, T. E., Moyes, C. D., and Tibbits, G. F. (2003) Am. J. Physiol. 284, C1176–C1184
  30. Robertson, S., and Potter. D. (1984) Methods Pharmacol. 5, 63–75
  31. Davis, J. P., Tikunova, S. B., Walsh, M. P., and Johnson, J. D. (1999) Biochemistry 38, 4235–4244[CrossRef][Medline] [Order article via Infotrieve]
  32. Dantzig, J. A., Goldman, Y. E., Millar, N. C., Lacktis, J., and Homsher, E. (1992) J. Physiol. 451, 247–278[Abstract/Free Full Text]
  33. Patton, C., Thompson, S., and Epel, D. (2004) Cell Calcium 35, 427–431[CrossRef][Medline] [Order article via Infotrieve]
  34. Koradi, R., Billeter, M., and Wuthrich, K. (1996) J. Mol. Graphics 14, 51–55[CrossRef][Medline] [Order article via Infotrieve]
  35. Hazard, A. L., Kohout, S. C., Stricker, N. L., Putkey, J. A., and Falke, J. J. (1998) Protein Sci. 7, 2451–2459[Medline] [Order article via Infotrieve]
  36. Falke, J. J., Drake, S. K., Hazard, A. L., and Peersen, O. B. (1994) Q. Rev. Biophys. 27, 219–290[Medline] [Order article via Infotrieve]
  37. Linse, S., and Forsen, S. (1995) Adv. Second Messenger Phosphoprotein Res. 30, 89–151[Medline] [Order article via Infotrieve]
  38. Wang, S., George, S. E., Davis, J. P., and Johnson, J. D. (1998) Biochemistry 37, 14539–14544[CrossRef][Medline] [Order article via Infotrieve]
  39. Black, D. J., Tikunova, S. B., Johnson, J. D., and Davis, J. P. (2000) Biochemistry 39, 13831–13837[CrossRef][Medline] [Order article via Infotrieve]
  40. Tikunova, S. B., Black, D. J., Johnson, J. D., and Davis, J. P. (2001) Biochemistry 40, 3348–3353[CrossRef][Medline] [Order article via Infotrieve]
  41. Browne, J. P., Strom, M., Martin, S. R., and Bayley, P. M. (1997) Biochemistry 36, 9550–9561[CrossRef][Medline] [Order article via Infotrieve]
  42. Davis, J. P., Rall, J. A., Alionte, C., and Tikunova, S. B. (2004) J. Biol. Chem. 279, 17348–17360[Abstract/Free Full Text]
  43. Black, D. J. (2001) Biophys. J. 80, 408 (abstr.)
  44. Fujimori, K., Sorenson, M., Herzberg, O., Moult, J., and Reinach, F. C. (1990) Nature 345, 182–184[CrossRef][Medline] [Order article via Infotrieve]
  45. Grabarek, Z., Tan, R.Y., Wang, J., Tao, T., and Gergely, J. (1990) Nature 345, 132–135[CrossRef][Medline] [Order article via Infotrieve]
  46. Tan, R.Y., Mabuchi, Y., and Grabarek, Z. (1996) J. Biol. Chem. 271, 7479–7483[Abstract/Free Full Text]
  47. Ababou, A., and Desjarlais, J. R. (2001) Protein Sci. 10, 301–312[CrossRef][Medline] [Order article via Infotrieve]
  48. Ababou, A., Shenvi, R. A., Desjarlais, J. R. (2001) Biochemistry 40, 12719–12726[CrossRef][Medline] [Order article via Infotrieve]
  49. Dong, W. J., Rosenfeld, S. S., Wang, C. K., Gordon, A. M., and Cheung, H. C. (1996) J. Biol. Chem. 271, 688–694[Abstract/Free Full Text]
  50. Tikunova, S. B., Davis, J. P., and Rall, J. A. (2004) Biophys. J. 86, 394 (abstr.)
  51. Solaro, R. J., and Shiner, J. S. (1976) Circ. Res. 39, 8–14[Abstract/Free Full Text]
  52. Best. P. M., Donaldson, S. K., and Kerrick, W. G. (1977). J. Physiol. 265, 1–17[Abstract/Free Full Text]
  53. Rupp, H. (1982) Adv. Myocardiol. 3, 455–466[Medline] [Order article via Infotrieve]
  54. Donaldson, S. K., Best, P. M., and Kerrick, G. L. (1978) J. Gen. Physiol. 71, 645–655[Abstract/Free Full Text]
  55. Allen, K., Xu, Y. Y., and Kerrick, W. G. (2000) J. Appl. Physiol. 88, 180–185[Abstract/Free Full Text]
  56. Johnson, J. D., Collins, J. H., Robertson, S. P., and Potter, J. D. (1980) J. Biol. Chem. 255, 9635–9640[Abstract/Free Full Text]
  57. Hongo, K., Konishi, M., and Kurihara, S. (1994) Jpn. J. Physiol. 44, 357–378[CrossRef][Medline] [Order article via Infotrieve]
  58. Ohki, S., Ikura, M., and Zhang, M. (1997) Biochemistry 36, 4309–4316[CrossRef][Medline] [Order article via Infotrieve]
  59. Andersson, M., Malmendal, A., Linse, S., Ivarsson, I., Forsen, S., and Svensson, L. A. (1997) Protein Sci. 6, 1139–1147[Medline] [Order article via Infotrieve]
  60. Malmendal, A., Evenas, J., Thulin, E., Gippert, G. P., Drakenberg, T., and Forsen, S. (1998) J. Biol. Chem. 273, 28994–29001[Abstract/Free Full Text]
  61. Sayle, R. A., and Milner-White, E. J. (1995) Trends Biochem. Sci. 20, 374–376[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
B. Liang, F. Chung, Y. Qu, D. Pavlov, T. E. Gillis, S. B. Tikunova, J. P. Davis, and G. F. Tibbits
Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca2+ binding and myofilament contractility
Physiol Genomics, April 1, 2008; 33(2): 257 - 266.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. P. Davis and S. B. Tikunova
Ca2+ exchange with troponin C and cardiac muscle dynamics
Cardiovasc Res, March 1, 2008; 77(4): 619 - 626.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
T. E. Gillis, C. R. Marshall, and G. F. Tibbits
Functional and evolutionary relationships of troponin C
Physiol Genomics, December 19, 2007; 32(1): 16 - 27.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Norman, J. A. Rall, S. B. Tikunova, and J. P. Davis
Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2580 - H2587.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. V. Gomes, G. Venkatraman, J. P. Davis, S. B. Tikunova, P. Engel, R. J. Solaro, and J. D. Potter
Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I: INSIGHTS INTO THE ROLE OF TROPONIN T ISOFORMS IN THE FETAL HEART
J. Biol. Chem., November 26, 2004; 279(48): 49579 - 49587.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/34/35341    most recent
M405413200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tikunova, S. B.
Right arrow Articles by Davis, J. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tikunova, S. B.
Right arrow Articles by Davis, J. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement