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J. Biol. Chem., Vol. 279, Issue 25, 26612-26618, June 18, 2004

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Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge^*

Aileen E. Boyd, Cristina S. Dunlop¶, Lilly Wong, Zoran Radi, Palmer Taylor||, and David A. Johnson**

From the Department of Pharmacology, University of California, La Jolla, California 92093-0636 and the ^**Division of Biomedical Sciences, University of California, Riverside, California 92521-0121

Received for publication, February 10, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys⁶⁹-Cys⁹⁶ loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Catalysis of the hydrolysis of acetylcholine by the serine hydrolase, acetylcholinesterase (AChE),¹ occurs at or near the diffusion limit (1, 2). The crystal structure of AChE reveals the enzyme active center to be at the base of a narrow, aromatic side chain lined gorge, some 18-20 Å in depth (3-5). How the substrate can rapidly traverse this tortuous route, acylate the active center serine with concomitant choline departure, and then deacylate with acetate departure in each catalytic cycle of 100 µs has been a source of puzzlement. A large dipole moment aligned with the gorge entry portal enhances diffusional ingress (6-8). Alternative portals have been proposed as routes for access or removal of H₂O, H⁺, or products (9-11); however, the finding that mutations in the vicinity of the proposed alternative portals do not alter steady-state catalytic parameters argues against a second access route being a rate-limiting step (12, 13).

Molecular dynamic simulations have pointed to flexibility and/or fluctuations in gating that may enhance accessibility to the active center (14, 15). A 10-ns molecular dynamic simulation of mouse AChE analyzed in terms of projections on the principal components suggests that collective motions on many time scales contribute to the opening of the gorge (16). Residues at the gorge opening and constriction point generally have larger correlation vectors pointing away from the gorge than do residues located peripheral to the gorge, suggesting large amplitude gorge opening motions.

Although molecular dynamic simulations support the existence of a conformationally active gorge, little experimental support exists. Analysis of the various published crystal structures of the apo and ligand-bound enzyme from various sources yield no indication that the gorge exists in "open" and "closed" states. On the other hand, Laue crystallography, with its diminished exposure time of x-ray radiation, has the potential to detect short lifetime intermediates in formation of AChE complexes (17). Also, we have reported that both active center and peripheral site inhibitors alter dramatically the emission maxima of acrylodan conjugated to mouse AChE cysteine mutants (18). Because many of the affected residues do not directly contact the bound ligand, the findings reveal ligand-dependent conformational changes in the Cys⁶⁹-Cys⁹⁶ -loop, which forms one of the walls of the active center gorge (18, 19). Additionally, we have found substantial internal flexibility at this -loop as well as changes in flexibility induced by ligand binding (20).

To extend our observations on the AChE conformational dynamics to additional areas around the active center gorge, we measured the effects of two inhibitors on the backbone flexibility of three surface sites across the active center gorge from the Cys⁶⁹-Cys⁹⁶ -loop using site-directed labeling in combination with time-resolved fluorescence anisotropy. Specifically, we chose three aligned sites for cysteine substitution and fluorescein conjugation, starting at the rim of the gorge (H287C), extending radially to an adjacent surface -helix (T249C), and then to the tip of a small and mobile -loop (A262C), some 20 Å away from the gorge rim (Fig. 1). Timeresolved fluorescence anisotropy yields information on the excited state of the fluorophore in the picosecond to nanosecond time frame (21), a time domain much shorter than the AChE catalytic cycle. With this approach we hoped to assess the basic characteristics of the motion at sites near the gorge but across from the Cys⁶⁹-Cys⁹⁶ -loop. We reasoned that if the opening motions were conformationally linked, ligand association, which would influence peptidyl backbone flexibility adjacent to the bound ligand, should also affect motion and flexibility parameters at more distal loci. The results reveal distinct modes of molecular motion in the individual regions examined. Also, the very limited effects of ligand binding upon anisotropy decay suggest uncoupled movements of the regions examined, a finding consistent with a model for transient gorge openings that is dominated by random segmental movements.

View larger version (98K): [in this window] [in a new window]   FIG. 1. Locations of the sites of cysteine substitution and fluorescein conjugation. Illustrated is a surface presentation of mouse AChE looking into the active center gorge (Protein Data Bank code 1KU6) with the Cys69-Cys96 -loop, highlighted in green, and the three sites of cysteine substitution and fluorescein conjugation (Thr249, Ala262, and His287), highlighted in yellow. The position of bound tacrine, shown in red, was derived from the crystallographic coordinates of the Torpedo californica AChE-tacrine complex (Protein Data Bank code 1ACJ [PDB] ) and overlaid into the mouse AChE structure.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzyme Preparation—Mutagenesis procedures to generate the cysteine-substituted mutants are described elsewhere (22). Briefly, cDNAs encoding a monomeric form of the mouse enzyme truncated at position 548 were placed in the mammalian expression vector, pcDNA3 (Invitrogen), and were subjected to PCR-mediated (QuikChange^TM, Stratagene) mutagenesis. Restriction enzyme analyses allowed detection of the mutations. To ensure the absence of spurious mutations, cassettes encompassing the mutated site were subcloned into pcDNA3 vectors that had not been exposed to the mutagenesis procedure, and their nucleotide sequences were verified by double stranded sequencing.

Human embryonic kidney (HEK 293) cells, purchased from American Type Culture Collection (Manassas, VA), were plated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at a density of 1.5 x 10⁶ cells per 10-cm dish, 24 h prior to transfection. Standard HEPES/calcium phosphate precipitation methods were used to apply 10 µg of plasmid DNA per plate to the cell monolayers. The next day, cells were rinsed with phosphate-buffered saline and supplied with serum-free media for continued growth (Ultraculture, Bio-Whitaker, Walkersville, MD). Large scale productions of mutant enzymes entailed creation of stable cell lines that exhibited G418 (Gemini Bio-Products, Woodland, CA) resistance following cotransfection with a neomycin resistance gene as described elsewhere (22, 23). Harvests of mutant AChE in serum-free media from confluent cells in three-tiered flasks (Nalge Nunc Int., Rochester, NY) typically continued for several weeks after which expression levels began to decline.

Affinity chromatography with trimethyl (m-aminophenyl)ammonium linked through a long tether arm to Sepharose CL-4B resin (Sigma) permitted one-step purification of AChE, both mutant and wild-type in amounts between 5 and 25 mg, as previously described (24-26). Purity was assessed by SDS-PAGE and by comparisons of specific activity with absorbance at 280 nm to measure protein concentration (₂₈₀ = 1.14 x 10⁵ M^-1 cm^-1 (27)).

Catalytic Activity—The catalytic activity of each unlabeled and labeled mutant was measured with the Ellman assay (28). K_m and K_ss (the dissociation constant of a ternary complex resulting in substrate inhibition or activation) were evaluated as described in previous kinetic schemes (23). The x intercept of a plot of the residual catalytic activity versus the concentration of the irreversible inhibitor 7-[[(methylethoxy)-phosphinyl]-oxyl]-1-methylquinolinium iodide yielded the enzyme concentration, and, in turn, k_cat (29).

Fluorescein Labeling—The mutant enzymes were pretreated with 0.25 mM dithiothreitol for 30 min at room temperature to ensure that all of the free cysteines were reduced, and free dithiothreitol was removed with a G-50 Sephadex spin column (Roche Diagnostics) equilibrated in 0.1 M sodium phosphate buffer, pH 7. FM and IAF were dissolved in dimethylformamide to make stock concentrations between 6 and 15 mM. The thioreactive probes, FM and IAF, at 100 times the enzyme concentration, were added to the enzyme solutions to achieve either a 3-fold molar excess in the case of the A262C mutant or a 20-fold molar excess in the case of the T249C and H287C mutants. Labeling was allowed to proceed for 12 h at 4 °C for the A262C mutant and for 2 h at 37 °C for the T249C and H287C mutants.

Unconjugated fluorescein derivatives were removed by gel-filtration with G-25 Sephadex (Amersham Biosciences), equilibrated with 0.1 M sodium phosphate buffer, pH 7. Parallel labeling reactions with wild-type mouse AChE were performed to assess nonspecific labeling. Stoichiometries of fluorescein-labeled mutants were estimated spectrophotometrically by substitution of the measured absorbance values at 280 nm (A₂₈₀) and 495 nm (A₄₉₅) into the following expression.

(Eq. 1)

Steady-state Emission—Steady-state emission spectra were measured at room temperature using a FluoroMax II spectrofluorometer (Jobin Yvon Inc., Edison, NJ).

Time-resolved Fluorescence Anisotropy—Emission anisotropy was determined by time-correlated single photon-counting measurements (30) with an IBH (Edinburgh, UK) 480-nm NanoLED^TM flash lamp run at 1 MHz and IBH model TBX-04 photon detector. The vertically [I_||(t)] and orthogonally [I(t)] polarized emission components were collected by exciting samples with vertically polarized light while orienting the emission polarizer (Polaroid HNPNB dichroic film) in either a vertical or orthogonal direction. Excitation and emission bands were selected with an Oriel 500-nm short-pass interference filter (catalog number 59876) and a Corning 3-68 cut on filter with a half-maximum transmission of 540 nm, respectively. Typically, 2 x 10⁴ peak counts were collected (in 1-2 min) when the emission polarizer was vertically oriented. The orthogonal emission decay profile was generated over the same time interval that was used to generate the vertical emission decay profile. Samples were held at 22 °C. To minimize convolution artifacts, flash lamp profiles were recorded by removing the emission filter and monitoring light scatter from a suspension of latex beads. The data analysis software corrected the wavelength-dependent temporal dispersion of the photoelectrons by the photomultiplier. The polarization bias (G) of the detection instrumentation was determined by measuring the integrated photon counts/6 x 10⁶ lamp flashes while the samples were excited with orthogonally polarized light and the emission was monitored with a polarizer oriented in the vertical and orthogonal directions (G = 1.028).

The emission anisotropy decay, r(t), given by the expression,

(Eq. 2)

and total emission decay, S(t), for a macroscopically isotropic sample,

(Eq. 3)

were deconvolved simultaneously from the individual polarized emission components expressed as,

(Eq. 4)

and the following.

(Eq. 5)

Thus, both I_||(t) and I(t) are determined by the same fitting functions, S(t) and r(t), and fitting parameters.

The fluorescence lifetimes for each sample were determined by initially generating a total emission decay profile from I_||(t) and I(t) with Equation 3 and then globally fitting I_||(t) and I(t) decay profiles to Equations 4 and 5 with the lifetime parameters fixed and with the following anisotropy decay expression.

(Eq. 6)

Here, r₀ is the amplitude of the anisotropy at time 0, f_xb is the fraction of the anisotropy decay associated with the fast decay processes, and is the rotational correlation time of the anisotropy decay. The subscripts fast and slow denote the fast and slow decay processes, respectively. A nonassociative model was assumed, indicating that the emission relaxation times are common to all the rotational correlation times. The fluorescence data were analyzed using the Globals software package developed at the Laboratory for Fluorescence Dynamics at the University of Illinois, Urbana-Champaign. Goodness of fit was evaluated from the values of the reduced and by visual inspection of the weighted-residual plots.

To better define the uncertainty of the measured slow, presumably whole body, rotational correlation times, a range (not a mean ± S.D.) of _slow values was determined for each data set that produced a reasonably acceptable fit to the anisotropy decay. Specifically, a unidimensional search procedure was performed that involved directed searches along the _slow parameter axis, not allowing other fitting parameters to vary, to find the minimum and maximum _slow values that raised the values by 5%.

Estimation of Stokes' Radius—The Stokes' radius of wild-type AChE was estimated by gel filtration with a Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with 0.1 M ammonium acetate buffer, pH 7.2. The elution volumes of wild-type AChE and proteins with known Stokes' radii were measured. The estimated value of the wild-type AChE Stokes' radius of wild-type AChE was read from a curve derived using the known Stokes' radii and the elution parameters of the standards, following the equation of Porath (31).

Molecular Dynamic Simulations of FM-H287C Mutant—A series of short, 3-ps molecular dynamic simulations were performed on the mouse AChE-fasciculin complex coordinates (Protein Data Bank code 1KU6) with FM attached to Cys²⁸⁷ to assess the effects of bound fasciculin on the torsional rotation of the succinimidylthioether (maleimide) tether arm connecting the fluorescein reporter group to the -carbonyl backbone. The hydrogens were added in InsightII prior to calculations, and the pH was set to 7.0. Sets of 21 molecular dynamic simulations were run with an SGI Octane computer (Silicongraphics, Inc., Mountain View, CA) using the Discover 2.9 module within the InsightII 2000.1 computer program (Accelrys, San Diego, CA), both in the presence and absence of fasciculin. With the exception of the 19 AChE residues (from Gln²⁷⁹ to Phe²⁹⁷) forming a surface loop, including FM attached to Cys²⁸⁷, all the atoms of mouse AChE and fasciculin were "frozen" during simulation. No water was present, the dielectric constant was set to 80, and the structure was not minimized prior to the subsequent molecular dynamic simulations. The protocol included initial equilibration of the system at 300 K, heating to 700 K, then slow cooling in 50-K increments back to 300 K, followed by minimization of the system.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Characterization of Unlabeled and Labeled Mutants—SDS-PAGE analysis of the labeled and unlabeled mutants revealed a single, wide band of silver staining migrating with the variably glycosylated wild-type mouse AChE. Comparison of the steady-state emission from labeled mutants and parallel, wild-type AChE showed 6-7% nonspecific labeling. Based upon spectrophotometric ratios, stoichiometries of labeling for the labeled mutants were: T249C, 0.2 ± 0.04; A262C, 0.4 ± 0.1; and H287C, 0.3 ± 0.06. The K_m, K_ss, and k_cat values for the cysteine mutants, the conjugated cysteine mutants, and wild-type AChE preparations were within experimental error of each other (data not shown). Because labeling was not stoichiometric, we also looked for a fractional component in the substrate concentration dependence curves that showed a different K_m, but were unable to detect two components in the curves. Moreover, titration with both tacrine and fasciculin produced virtually complete inhibition of catalytic activity of the labeled mutants with similar K_I values as observed for the wild-type AChE. Together, these findings indicate that labeled mutants fold correctly despite cysteine substitution and reporter group conjugation at these positions.

Fluorescence Emission—The uncorrected emission (517 ± 1 nm) and excitation (494 ± 1 nm) maxima for the conjugated enzymes are typical of fluorescein in an aqueous environment. Neither fasciculin nor tacrine had a significant effect upon the excitation or emission spectra of either the T249C or A262C conjugates; however, fasciculin red shifted slightly (2-3 nm) the emission spectrum and enhanced the apparent quantum yield by 12% of the IAF-labeled H287C mutant, indicating a ligand-induced change of the microenvironment around the reporter group attached to the H287C site of conjugation (data not shown).

Estimation of Rotational Correlation Times—From the gel filtration measurements, wild-type AChE was estimated to have a Stokes' radius of 51 Å. Substituting this value into the Stokes-Einstein equation yielded a rotational correlation time of 128 ns.

Time-resolved Fluorescence Anisotropy of FM Conjugates—For brevity, the emission and the anisotropy decay profiles of only the FM-T249C conjugate are illustrated (Fig. 2). The time course of emission decay of each mutant examined was well fit to a biexponential decay model. For simplicity, only the geometric averaged excited state lifetimes are summarized (Table I) and ranged between 4.11 and 4.26 ns. The actual range of short and long wavelength emission lifetimes for the various nonliganded mutants were 4.26-4.82 and 2.98-4.01 ns, respectively. The amplitudes of the longer lifetime components ranged between 31 and 87% of the total decay amplitude (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Emission and anisotropy decay of the FM-T249C mouse AChE mutant. The upper panel shows the parallel () and perpendicular (+) emission decays (single data points), the fit of these data points to a single exponential decay equation (smooth lines), and the flash lamp profile (dashed line). The lower panel shows the time-resolved anisotropy decay (+), and a solid line through these points that was generated with the best-fit parameters (Table I) for a double exponential nonassociative decay model (Equation 6). The concentration of the labeled mutant was 150 nM.

Visual comparison of the time-resolved anisotropy decays of the three mutants (Fig. 3) shows that the fluorescein attached to A262C and H287C mutants reorients (depolarizes) more rapidly than the fluorescein attached to the T249C mutant, but with similar time 0 anisotropy values. Quantitatively, these similarities and differences were primarily observed in the fractional amplitude of the measurable fast decay processes (f_xb). The f_xb values for the FM-A262C and FM-H287C conjugates represent about half of the resolvable anisotropy decay (0.47 and 0.50, respectively), but only a third (0.32) of the FM-T249C mutant. The values for the time 0 anisotropy (r₀; range 0.29-0.30) and the fast rotational correlation time (_fast; range 0.8-1.2 ns) were essentially the same for all the mutants studied. Taken together, these results indicate a high level of -carbon flexibility around each residue examined, although the Thr²⁴⁹ residue is significantly less flexible than the Ala²⁶² and His²⁸⁷ residues.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Anisotropy decays of FM-T249C, FM-A262C, and FM-H287C mouse AChE mutants. Shown are the time-resolved anisotropy decays (single data points), and the solid lines through these points that were generated with the best-fit parameters (Table I) for a double exponential nonassociative decay model (Equation 6). The peak of the flash lamp profile arbitrarily defined the zero time point. The concentrations of FM-T249C (+), FM-A262C (), and FM-H287C () were 150, 190, and 135 nM, respectively.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Effect of fasciculin and tacrine on the anisotropy decay of the FM- and IAF-labeled H287C mutants. The time-resolved anisotropy decay (single data points) and the solid lines through these points were generated with the best-fit parameters (Table II) for a double exponential nonassociative decay model (Equation 6). Data points represent enzyme without inhibitors (), with tacrine (+), or with fasciculin (). The peak of the flash lamp profile arbitrarily defined the zero time point. The concentrations of FM-H287C, IAFH287C, fasciculin, and tacrine were 135 nM, 145 nM, 4.7 µM, and 40 µM, respectively.

View larger version (89K): [in this window] [in a new window]   FIG. 5. Molecular dynamic simulations of the effect of fasciculin binding on the torsional movements of Cys287 and attached FM. Twenty-one 3-ps simulations were performed on the x-ray coordinates of the mouse AChE-fasciculin complex with FM attached to Cys287 in the presence (A and C) and absence (B and D) of fasciculin as described under "Experimental Procedures." Only the residues between Gln279 and Phe297 and attached FM were allowed to move; the rest of mouse AChE and fasciculin atoms were "frozen" for the simulations. The positions of the FM bonds and the Gln279 to Phe297 backbone are shown in a different color for each simulation. Transparent Connolly surfaces of mouse AChE and fasciculin are shown in gray and brown, respectively. In panels C and D, the views of the positions of the FM bonds and the Gln279 to Phe297 backbone are rotated by 60° around the vertical axis and enlarged from the panel (A and B) above.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Time-resolved fluorescence anisotropy enables one to monitor directly reorientation of a fluorophore in the time frame of several excited state lifetimes. When a fluorophore is tethered to the macromolecule, depolarization is achieved by multiple relaxation modes that most likely occur in distinct time frames. Torsional motion around the tether arm, if unhindered, will occur in a subnanosecond time frame, not resolvable with commercial instruments. Restriction of motion around the tether will diminish the amplitude and slow the decay rate of this phase. Segmental motion of a domain within the molecule containing the fluorophore will give rise to a fast, but usually measurable depolarization; the rate and amplitude of which will depend upon the mass of the domain, the extent of the angular excursion, and the orientation of the absorbance/emission transition moments relative to the diffusion cone angle. Finally, global motion of the entire protein where the fluorophore is affixed to the macromolecule will in most cases produce the slowest rate of anisotropy decay. The latter will be governed by the multiple rotational correlation times of the macromolecule, which are not resolvable unless the molecule is highly dimensionally asymmetric.

Decay of Anisotropy of the FM Conjugates—The structure of the small -loop bounded by the disulfide bond at Cys²⁵⁷ and Cys²⁷² is not well resolved in AChE crystal structures unless the loop is stabilized by interaction with the symmetry related molecule (34). The large thermal or B factors for this region in the crystal structures suggest that the tip of this disulfide loop is either highly flexible or experiences static disorder in the crystal (35). Indeed, when FM is conjugated to A262C, the amplitudes of the very fast (r_i-r₀) and fast (r₀·f_xb) decay phases represent 17 and 39% of the total anisotropy decay, where r_i is the fundamental anisotropy of the fluorophore. Hence, independent motions of the fluorescein tether arm through torsional motion (r_i-r₀) and flexible domain motion (r₀-f_xb) in the AChE molecule are the dominant modes of anisotropy decay at the 262 position.

When fluorescein is attached to the H287C mutant, we note similar amplitudes for the very rapid and rapid decay phases, 14 and 43%. Again, the major mode of depolarization of the fluorescein conjugated at position 287 occurs by modes other than the global rotation of the molecule. The average crystallographic B factors for the -carbon chain atoms of residue His²⁸⁷ are far smaller than that seen for residue 262 (Table III). The disparity between B factors at the 287 and 262 positions, in the face of similar large components of rapid depolarization of the attached fluorescein, suggests either that His²⁸⁷ is stabilized in the crystalline state or that the large average B factors associated with Ala²⁶² arise from a equilibrium (i.e. static disorder) between multiple positions of the small -loop in the crystal structure. Stabilization of His²⁸⁷ by crystallization forces could be part of the process that favors a closed gorge state in the crystal and explains why no open gorge crystal structures of AChE have been observed.

Influence of Ligand Binding on the Anisotropy Decay Parameters—To examine whether ligand binding influences anisotropy decay and hence dynamic parameters at various locations near the active center gorge, we initially selected two ligands whose complexes with AChE have been determined crystallographically. Tacrine binds at the active center at the base of the gorge with its aromatic ring system stacked between the indole moiety of Trp⁸⁴ and the phenyl ring of Phe³³⁰ in Torpedo AChE (36) corresponding to Trp 86 and Tyr 337 of mouse AChE. In fact, the latter residue shows a conformational movement associated with tacrine binding causing a parallel stacking of the tacrine ring between the two aromatic side chains. By contrast, fasciculin, a peptide of 61 amino acids, associates at the mouth of the gorge greatly limiting ligand access to the catalytic center (4, 37). The AChE crystal structure shows that His²⁸⁷ approaches a van der Waals distance to the bound fasciculin residues.

The binding of tacrine, despite it being inhibitory to all of the conjugated enzymes, has no effect on the lifetime or anisotropy decay parameters for fluorescein conjugated at positions 249, 262, and 287. Hence, this result would indicate that ligand binding does not cause a global conformation change that would be evident at these three disparate locations on the macromolecule. However, this finding does not preclude ligands inducing changes in conformation and side chain mobility in regions of the AChE molecule that are not reflected in our three labeling positions (11, 18, 19, 38).

Fasciculin, which binds with a K_d for the wild-type enzyme in the range of 4 pM (33) and whose crystal structure shows a van der Waals peptide to protein contact area of 1100 Ų (4), also does not affect the anisotropy decay parameters at positions 262 and 249. However, conjugated fluorescein at position 287 shows a small change in the Stokes' shift and emission lifetime, and a major change in the decay of anisotropy. The reduction of the amplitudes of the rapid decay phases and the slowing of the rapid decay phase likely reflect a small torsional restraint on the tethered fluorescein and a stabilization of movement of the loop encompassing residue 287 produced by the bound fasciculin.

The differential influence of fasciculin on the anisotropy decay of three fluorophores positioned in distinct locations on the AChE molecule, again, indicates that this ligand does not induce global changes in the conformational dynamics of the enzyme. Based on the limited number of sites examined, fasciculin appears to have stabilized a local domain of the molecule reducing the intrinsic flexibility in the region around His²⁸⁷ and probably in neighboring areas of direct contact.

Implications—These results combined with our previous analysis of the segmental dynamics of three sites on the Cys⁶⁹-Cys⁹⁶ -loop (20) paint a consistent picture of AChE with conformationally active surface backbone elements whose movements are poorly coupled to one another. Such a conformationally dynamic structure supports a model for transient gorge openings that is dominated by random segmental movements.

Also, the above studies further demonstrate the potential of time-resolved studies of fluorescence anisotropy to examine molecular motion in distinct regions of a molecule and its ligand-associated complexes whose overall structural template has been delineated through x-ray crystallography. The principal advantages of the technique stem from measurements conducted in solution and under conditions simulating ligand binding in situ. Cysteine substitution mutagenesis enables one to select individual side chains in strategic regions of the molecule and examine each locus in a systematic fashion with multiple fluorophores. The individual fluorophores selected for conjugation can differ in lifetime, spectroscopic parameters, and capacity to modify the substituted cysteines. Hence, a comprehensive analysis of time-resolved fluorescence typically requires analysis of a variety of residue positions often with more than a single probe. This presents a particular challenge for larger extracellular proteins because their production usually requires eukaryotic expression systems that typically yield limited quantities of protein. Also, glycosylation and intrinsic disulfide bonds characteristic of extracellular proteins may add further complexity to achieving appropriate expression. Our study establishes that the residue-directed fluorophore approach to labeling can yield valuable information even when the macromolecule is a large glycosylated protein.

In summary, we have extended our previous study of the conformational dynamics of the AChE Cys⁶⁹-Cys⁹⁶ -loop, a segment that forms the outer wall of the active center gorge, to include three additional sites positioned roughly in a line that starts at the rim of the gorge across from the -loop and projecting radially 20 Å away from the gorge. Similar to our previous study, site-directed labeling in conjunction with time-resolved fluorescence anisotropy was utilized. The results reveal both distinct modes of molecular motion as well as a high degree of backbone flexibility. Additionally, there appears to be limited coupling of the conformational fluctuations between the sites examined, because ligand binding only affected the one site that was in close proximity to the bound inhibitor. This indicates that ligand association did not produce global changes in the conformational flexibility of the enzyme. Moreover, these results combined with our previous analysis of the conformational dynamics of the Cys⁶⁹-Cys⁹⁶ -loop add support to the view that the area around the active center gorge rim is conformationally active and is consistent with a model for transient gorge openings that are dominated by random segmental movements.

	FOOTNOTES

 
^* This work was supported in part by United States Public Health Services Grants R37-GM18360 and P42-E10337, Department of Army Medical Defense Grant 17C-951-5027, and a University of California Intercampus Grant. 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.

Supported by National Institutes of Health Training Grant GM07752.

^¶ Present address: Wayne State University School of Medicine, Detroit, MI 48201.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, University of California, La Jolla, CA 92093-0636. Tel.: 858-534-4028; Fax: 858-534-8248; E-mail: pwtaylor{at}ucsd.edu.

¹ The abbreviations used are: AChE, acetylcholinesterase; FM, fluorescein 5-maleimide; IAF, 5-iodoacetamidofluorescein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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