Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M506753200 on August 31, 2005

J. Biol. Chem., Vol. 280, Issue 44, 37236-37245, November 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/44/37236    most recent
M506753200v1
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 Burgess, B. R.
Right arrow Articles by Garboczi, D. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burgess, B. R.
Right arrow Articles by Garboczi, D. N.
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?

Dissection of Merozoite Surface Protein 3, a Representative of a Family of Plasmodium falciparum Surface Proteins, Reveals an Oligomeric and Highly Elongated Molecule*{boxs}

Brandt R. Burgess{ddagger}, Peter Schuck§, and David N. Garboczi{ddagger}1

From the {ddagger}Structural Biology Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852 and the §Division of Bioengineering and Physical Science, Office of the Director, Office of Research Services, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, June 21, 2005 , and in revised form, August 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of {alpha}-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 µM. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f0 > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmodium falciparum, the parasitic agent that causes most cases of fatal malaria, is estimated to infect over 500 million people annually (1), causing 1-3 million deaths among young children in sub-Saharan Africa. The rise of parasite resistance to currently available drugs has made malaria treatment difficult. Vaccines have great potential as lower cost alternatives to widespread drug treatment, since they may provide long lived protection from disease. The P. falciparum blood stage surface protein MSP32 (2, 3) is a vaccine candidate, since it is known to be a target of the immune response. Immunizations with several forms of recombinant MSP3 were each shown to protect monkeys against parasite challenge (4, 5). MSP3-specific antibodies in the presence of monocytes mediated killing of parasites in an antibody-dependent cellular inhibition assay (6). Homologs to MSP3 have been identified in several Plasmodium species (7-9), indicating that the molecule plays a conserved role in blood stage parasites.

The P. falciparum MSP3 sequence predicts several domains (Fig. 1): a central domain that includes three blocks of imperfect Ala heptad repeats of the sequence pattern Ala-X-X-Ala-X-X-X, a second central region rich in Glu residues, and a C-terminal leucine zipper-like domain (2, 3). Secondary structure algorithms predict MSP3 to be largely {alpha}-helical, especially in the Ala heptad repeat region that is predicted to form a coiled coil. A 38-residue peptide from the first of the three blocks of Ala heptad repeats was {alpha}-helical as determined by NMR (10).

MSP3 associates with the merozoite surface, although it does not have a recognizable transmembrane region or a signal sequence for a glycosylphosphatidyl inositol anchor (2, 3). The homologous P. falciparum merozoite surface proteins MSP6 (11, 12) and MSP11 (13) also contain Glu-rich and C-terminal leucine zipper-like domains and, together with MSP3, may identify a new family of proteins. MSP6 has been shown to interact, possibly through its C-terminal region, with the MSP1 and MSP7 complex (11). A similar interaction between the C-terminal region of MSP3 and another surface protein may be how MSP3 associates with the merozoite surface. There is evidence that suggests that MSP3 may interact with the cysteine protease ABRA (acid-base rich antigen) in vivo (14).

To gain a better understanding of the structure of MSP3, we performed chemical cross-linking, analytical ultracentrifugation (sedimentation equilibrium and sedimentation velocity), and CD experiments on various recombinant MSP3 polypeptides. Our results show that 1) the full-length recombinant MSP3 has high percentages of {alpha}-helix and random coil; 2) full-length MSP3 forms both dimers and tetramers in solution, with the tetramer apparently formed by an "end-to-end" arrangement of two dimers; 3) the oligomerization domain resides in the 60 residues at the C terminus of the full-length protein; 4) the MSP3 dimer and tetramer are highly elongated molecules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of the MSP3 Expression Plasmids—The gene encoding the K1 isolate of P. falciparum MSP3 (3), without the DNA encoding for the 19-residue signal sequence at the N terminus, was codon-optimized for expression in Escherichia coli (Bionexus, Inc., Oakland, CA). The full-length synthetic MSP3 gene was cloned into the T7 expression vector pLM1 (15). A His6 tag was added to the N terminus, and Tyr223 was mutated to Ala (Y223A) to avoid protease cleavage. The c308 construct was made by deleting residues 309-362 from the original non His6-tagged full-length MSP3, and a C-terminal His6 tag was added to block observed C-terminal degradation. The CC construct was made by deleting residues 1-62 and 224-362. The n224 construct was made by deleting residues 1-223. The LZ construct was made by deleting residues 1-305 and placing a His6 tag at the N terminus to allow purification of this small polypeptide. All deletions or insertions were derived from the full-length MSP3 gene in the pLM1 vector via standard mutagenesis procedures (QuikChange; Stratagene, La Jolla, CA). DNA sequencing of the entire coding region confirmed each gene sequence.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1.
The P. falciparum MSP3 protein sequence. A, shown here is the amino acid sequence of the full-length MSP3 minus the 19 residues of the N-terminal secretory signal sequence. Residues highlighted by colors are the three blocks of Ala-X-X-Ala-X-X-X heptad repeats (red), the glutamic acid-rich region (blue), and the putative leucine zipper-like domain (green). The N-terminal methionine is from the expression construct. B, P. falciparum MSP3 constructs produced for these experiments (coloring scheme same as in A). Labeled residues denote sequences defining the beginning and the end of the construct. Asterisks denote locations of His6 tags on the full-length, c308, and LZ proteins.

 
Protein Expression—One-liter cultures of BL21(DE3) cells containing the appropriate MSP3 expression plasmid were grown to an optical density of 0.8 at 600 nm, and protein expression was induced by the addition of 1 mM isopropyl-thiogalactoside. After 3.5 h, cells were harvested by centrifugation at 8,000 rpm (SLA-1500 rotor) for 10 min at 4 °C. The cells were washed with 20 mM Tris-HCl, pH 8.0, 2 mM NaEDTA, and 200 mM NaCl and pelleted. The cell pellets were resuspended in 50 ml of 50 mM Tris-HCl, pH 8.0, 5 mM NaEDTA, 2 mM dithiothreitol (DTT), 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 100 µg/ml lysozyme, and one Complete Protease inhibitor tablet (Roche Applied Science) per liter of culture. DTT (2 mM) was used to keep the single cysteine at residue 120 reduced throughout the purification procedure. The cells were subjected to two cycles of freeze/thaw in the presence of MgCl2 and DNase at 10 mM and 20 µg/ml, respectively. The supernatant containing soluble MSP3 protein was cleared of insoluble material by centrifugation at 12,000 rpm (SS-34 rotor) for 30 min. The cleared supernatant was stored at -80 °C.

Purification of the MSP3 Polypeptides—The cleared supernatant containing the full-length MSP3 was thawed and then heat-treated at 80 °C in 25-ml aliquots in sterile 50-ml conical Falcon tubes for 25 min. Insoluble material that was present after heat treatment was pelleted. Due to their inherent stability, most coiled coil proteins remain soluble at high temperature (16), whereas many other proteins denature and/or aggregate and precipitate out of solution. The full-length and other MSP3 polypeptides remained soluble in the supernatant. Samples of heated and nonheated cleared supernatants for these proteins were fractionated on a size exclusion column (HiLoad 16/60; Superdex 200; Amersham Biosciences) equilibrated in phosphate-buffered saline (PBS) (1.7 mM KH2PO4, 5 mM Na2HPO4, 150 mM NaCl) with 2 mM DTT. The MSP3 polypeptides exhibited the same elution volumes whether they were heated or not, confirming that the native fold or oligomeric state was not significantly altered after the heating and cooling steps (supplemental Fig. S1A). Compared with not heating, use of the heating step resulted in the higher level of purity required for structural studies.

The cleared supernatant following the heat step was loaded on an anion exchange column (HiLoad 16/10 High Performance Q-Sepharose column; Amersham Biosciences) equilibrated in 50 mM MES-NaOH, pH 5.5, and 2 mM DTT. Full-length MSP3 was eluted with a 0-600 mM NaCl gradient. Fractions containing the protein were pooled, concentrated (Centriprep 10,000), and then chromatographed on a size exclusion column (Superdex 200) in PBS with 2 mM DTT. The fractions from a single resolved peak that contained the protein were pooled and concentrated. Next, crystalline urea was added to a final concentration of 8 M. The denatured sample was then loaded on a nickel nitrilotriacetic acid (Ni2+-NTA) column (Qiagen, Valencia, CA) previously equilibrated in PBS, 2 mM DTT, and 8 M urea and was followed by a wash of 20 column volumes of the same buffer. The protein was eluted by the addition of 300 mM imidazole-HCl and was dialyzed (molecular weight cut-off of 10,000) at 4 °C against three changes (100x volume of protein sample) of PBS and 4 mM DTT over 24 h. Following dialysis, the protein was concentrated and chromatographed three times in succession on the size exclusion column in PBS and 2 mM DTT to assure an adequate separation of soluble aggregate and the properly refolded His-tagged full-length MSP3 protein.

The c308 protein was purified as above, except that the Ni2+-NTA column was run in PBS and 2 mM DTT, without urea. The CC protein was purified using the same procedure as the full-length MSP3 except that the buffer used during the anion exchange step was 50 mM HCl, pH 8.0, Tris- and no Ni2+-NTA column was used. The n224 protein was purified with the same procedure as the full-length MSP3 except that no Ni2+-NTA column was used. The LZ protein was purified the using the same procedure as the full-length MSP3 except that no anion exchange step was used, and the Ni2+-NTA column was used under native conditions without urea.

Amino acid sequencing and ion spray mass spectrometry confirmed the N-terminal sequence and mass of each polypeptide, respectively. Mass spectrometry also confirmed that the single cysteine residue of MSP3 was in a reduced state in all polypeptides. Protein concentrations were determined by absorbance at 280 nm. Coomassie Brilliant Blue-stained SDS-PAGE indicated that the purities of the proteins were greater than 95%. All proteins were stored at -80 °C.

Chemical Cross-linking—All MSP3 proteins were analyzed by primary amine cross-linking using ethylene glycol bis-(succinimidylsuccinate) (EGS) (Pierce) prepared as a 25 mM stock solution in dimethyl sulfoxide. All proteins, at 1 and 0.2 mg/ml in 0.5x PBS and 1 mM DTT, were mixed with EGS to make a 2-fold molar excess of EGS over the concentration of Lys residues and were incubated at 23 °C. At each time point, an aliquot was taken from the reaction mixture and quenched by the addition of 1 M Tris-HCl, pH 8.0, to a final concentration of 100 mM. The samples were then mixed with an equal volume of SDS-PAGE loading buffer containing 100 mM Tris-HCl, pH 8, 5% SDS, 0.3% bromphenol blue, and 10 mM tris(2-carboxyethyl)phosphine and loaded without heating on a 10% Bis-Tris polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue and subsequently destained for analysis.

Circular Dichroism—CD measurements were recorded using a Jasco J-810 spectropolarimeter with a Neslab RTE-111 water bath to maintain a constant temperature during analysis. All proteins were diluted to 0.2 mg/ml in PBS (final concentration of DTT was ≤0.2 mM) and loaded into a 2-mm quartz cuvette. The blank in all cases was PBS buffer and 0.2 mM DTT. Each final spectrum was an average of three sequential scans. For the thermal denaturation of the full-length protein, spectra were measured at 10 °C increments from 5 to 85 °C (the high temperature limit of the water bath). Before each reading, the sample cuvette equilibrated at each temperature for 10 min. No base-line shift due to the oxidation of DTT was observed throughout the control experiment performed with PBS buffer and 0.2 mM DTT alone. Temperature readings taken within the cuvette holder agreed with the temperature of the water bath to within 2 °C. All CD data were converted to mean residue ellipticity (degrees cm2 dmol-1). The CDPro software package (lamar.colostate.edu/~sreeram/CDPro) was used to estimate the amount of secondary structure from the CD spectra and was based on the reference set SP37A (ibasis 5) (17). CDPro and all software used in these studies are available without charge.

Sedimentation Equilibrium—Studies were performed in an Optima XL-I/A analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) at rotor temperatures of 8 and 10 °C and speeds between 14,000 and 25,000 rpm, using the long column technique (18, 19) and protocols (20) previously described. In brief, protein at concentrations between 0.1 and 1.5 mg/ml in PBS (150-180 µl) was loaded into charcoal-filled Epon double sector centerpieces, and absorbance distributions were recorded in 0.001-cm radial intervals, averaging 20 data samples for each point. In order to detect a wide concentration range, scans were taken simultaneously at wavelengths of 230, 250, and 280 nm. Attainment of equilibrium was verified after 48-72 h by comparing sequential scans taken in 6-h intervals using the program WINMATCH (kindly provided by Drs. David Yphantis and Jeffrey Lary, University of Connecticut). Protein partial specific volumes were calculated from amino acid composition using the software SEDNTERP (kindly provided by Dr. John Philo, Alliance Protein Laboratories), leading to values at 4 °C of 0.714 ml/g for the full-length protein, 0.712 ml/g for c308, 0.715 ml/g for CC, 0.702 ml/g for n224, and 0.728 ml/g for LZ.

Global least-squares modeling of absorbance profiles at multiple wavelengths, concentrations, and/or rotor speeds was performed with the software SEDPHAT (19), based on the well known superpositions of the Boltzmann distributions of ideal species in a centrifugal field (21) using several models: a single stable species, a single species with trace impurity, and a self-associating system (19), which can be expressed generally as follows,

(Eq. 1)

with

(Eq. 2)

where a{lambda} is the measured absorbance at wavelength {lambda}, r denotes the distance from the center of rotation, r0 is a reference radius, ci and cj are the concentrations of an i-mer and j-mer, respectively, Kij is an association constant from i-mer to j-mer, c*i and c*j are local concentrations of species not participating in the association, d is the optical path length, {epsilon}{lambda} is the calculated extinction coefficient, M1 and are the protein molar mass and partial specific volume, {rho} is the solvent density, {omega} is the rotor angular velocity, R is the gas constant, and T is the absolute temperature. The global fit was performed using implicit global mass conservation constraints, relating the total mass of protein in solution among the different rotor speeds (19), and expressing the concentration of material not participating in the association as constant fractions of the total loaded protein in each cell. The bottom position of the solution column was a fitted parameter (19). The results of the sedimentation equilibrium analysis were confirmed independently by sedimentation velocity and dynamic light scattering experiments.

Sedimentation Velocity—Studies were conducted following the experimental protocol previously reported (20). Briefly, 400 µl of protein dissolved in PBS and 400 µl of PBS were loaded into the sample and reference chambers, respectively, of charcoal-filled Epon double sector centerpieces. Following a 1-h temperature equilibration period at 20 °C at rest, the rotor was accelerated to 50,000 rpm, and refractive index profiles were recorded in 1-min intervals with the Rayleigh interference optical system, covering the sedimentation process from initially partial depletion at the meniscus until the sedimentation boundary migrated outside the observable radial range.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2.
Size exclusion chromatography of the MSP3 polypeptides. The chromatograms for the full-length (FL), c308, CC, n224, and standard proteins are plotted on the same x axis and offset in the y axis for clarity. MSP3 polypeptides and their calculated molecular weights are denoted on each chromatogram. The elution volumes for the LZ HMW and LZ LMW peaks (asterisks) were determined by polyacrylamide gel analysis of column fractions. The molecular mass standards in kDa shown at the bottom are as follows: thyroglobulin (670), bovine {gamma}-globulin (158), chicken ovalbumin (44), equine myoglobin (17), and vitamin B-12 (1.4) (Bio-Rad). The flow rate was 1 ml/min.

 
Data were modeled as a superposition of Lamm equation solutions, c(s), with the software SEDFIT (22). The time- and radius-dependent fringe displacement data a(r,t) were modeled as a superposition,

(Eq. 3)

of Lamm equation solutions,

(Eq. 4)

(with s and D denoting the sedimentation and diffusion coefficient, respectively), using the hydrodynamic scaling law,

(Eq. 5)

with {eta} denoting the solvent viscosity and k the Boltzmann constant, and Fw a weight-average frictional ratio of all species (23). In Equation 3, aTI(r) and aRI(t) are the systematic signal contributions from time- and radius-invariant noise, calculated algebraically (24). The sedimentation coefficient distribution c(s) was calculated using maximum entropy regularization at a confidence level of p = 0.7 and optimizing Fw and the meniscus position of the solution column by nonlinear regression. Fits were obtained with root mean square deviations between 0.005 and 0.008 fringes.

The s value of the molecules was determined by integration of the main peaks of c(s). Alternatively, for some constructs, global modeling of data obtained at different loading concentrations was performed with SEDPHAT using a model for multiple discrete species or using coupled Lamm equation solutions for kinetically controlled self-association (25). For each construct, the hydrodynamic frictional ratio (f/f0) was determined with SEDNTERP by combining the measured s value with the molar mass calculated from the amino acid sequence and the oligomeric state observed in sedimentation equilibrium. The determined frictional ratios (f/f0) are molecular constants that include the contributions from hydration. Using estimates of hydration based on the amino acid sequence (26), this frictional ratio can be divided into a hydration and a shape factor (e.g. see Refs. 27 and 28), from which dimensions of ellipsoid models were derived. These calculations were performed with SEDNTERP.

Prediction of Secondary Structure—The SOPMA (29), DSC (30), and PREDATOR (31) algorithms were used to predict the secondary structure content of the full-length MSP3 protein to be 55-68% {alpha}-helix, less than 10% {beta}-sheet, and about 33% random coil.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Size Exclusion Chromatography of the Recombinant MSP3 Polypeptides—During purification, the full-length MSP3 polypeptide (42 kDa from sequence) exhibited an unexpected mobility on a size exclusion column, eluting at a volume corresponding to a molecular mass in the range of 400 to 500 kDa as compared with globular standards (Fig. 2). Since the purification procedure includes an 80 °C heat treatment, we confirmed that the full-length protein, purified without the heating step, also eluted on the same column at a size of 400-500 kDa (supplemental Fig. S1A). This result indicated that the heat treatment was not causing the atypical mobility and suggested that full-length MSP3 may be an oligomer and/or an elongated molecule. To address the causes of the unusual size of MSP3, we produced several shorter polypeptides containing subsets of the predicted domains of the full-length polypeptide (Fig. 1).

During size exclusion chromatography, the c308, n224, and LZ polypeptides also eluted at volumes corresponding to molecular weights that were several times larger than their predicted weights (Fig. 2). The c308 polypeptide (36 kDa from sequence) eluted at a size of about 110 kDa. The n224 polypeptide (16 kDa from sequence) eluted in a broad peak at about 400 kDa and larger than c308. The LZ peptide (8 kDa from sequence) also eluted abnormally fast, and polyacrylamide gel analysis showed that the peptide eluted as two peaks at 70 and 74 ml, eluting before the 44-kDa standard (Fig. 2). The two LZ peaks were carefully fractionated, pooled, and chromatographed again. Again, two peaks appeared, each at the same elution volume, leading us to conclude that these two peaks were stable species and were probably different oligomeric states of the polypeptide. The LZ polypeptides in the two peaks were designated as LZ high molecular weight (LZ HMW) and LZ low molecular weight (LZ LMW). The CC polypeptide (18 kDa from sequence) eluted near the predicted volume and thus may be more globular than the rest of the protein, since this sequence is predicted to form an intramolecular coiled coil (2, 3, 10).



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 3.
Chemical cross-linking of the MSP3 polypeptides. Cross-linking samples were taken at 5 min (5'), 15 min (15'), and 30 min (30'). No cross-linker was present in the 0 min sample (0'). After stopping the reaction with Tris-HCl, samples were electrophoresed on reducing 10% Bis-Tris SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Apparent molecular masses in kDa (far left) are those of the standard molecular mass marker proteins loaded at the left of each gel.

 
Chemical Cross-linking—To gain evidence concerning the oligomeric state of the various MSP3 polypeptides, they were subjected to chemical cross-linking with the lysine-specific cross-linker EGS at two protein concentrations, 1 mg/ml (Fig. 3) and 0.2 mg/ml (not shown). Without cross-linking, the full-length MSP3 (42 kDa from sequence) migrates at an apparent molecular mass of 55 kDa when compared with standards, probably due to its high content of charged amino acid residues (Fig. 3, time = 0 min). Upon incubation of full-length MSP3 with the cross-linker (Fig. 3, top left), bands corresponding to sizes of dimers, trimers, and tetramers were observed at the earliest time point of 5 min. The higher order bands became darker at 15 min and were darkest at 30 min as more protein was cross-linked and shifted higher in the gel. The ladder of cross-linked bands stops distinctly at the tetramer position without detectable higher order oligomers. Cross-linking results at both protein concentrations were similar, although not as much protein was shifted to the higher molecular weight bands at 0.2 mg/ml, suggesting a concentration effect on oligomerization between 0.2 mg/ml (5 µM) and 1.0 mg/ml (24 µM).

To investigate if the leucine zipper-like domain of the full-length protein is responsible for the observed oligomerization, the c308 polypeptide lacking that domain was treated with cross-linker (Fig. 3, top right panel). The cross-linking did not produce oligomers, since only a trace amount of protein shifted to a higher position on the gel with no increase over the 30-min time course. Since the c308 protein does not cross-link into higher order bands, the oligomerization domain probably resides in the C-terminal 60 residues of MSP3 that are deleted in the c308 polypeptide. Using full-length protein and c308 that were not heated during preparation yielded cross-linking patterns very similar to those of the heat-treated proteins as seen by gel analysis (Fig S1B).

Gel analysis of the cross-linking of the CC polypeptide containing the Ala heptad repeats showed that the mobility of the EGS-treated protein at 30 min was similar to that of the untreated protein at 0 min (Fig. 3, middle left). Whereas there was no detectable formation of higher order covalently cross-linked oligomers, the CC protein bands became fuzzy and migrated slightly faster. This occurred with the c308 molecule as well (Fig. 3, top right) and is probably due to intramolecular cross-linking that makes the molecules become more compact and migrate faster in the gel. The CC domain appears to be monomeric in solution at concentrations up to 1 mg/ml (56 µM).

When n224 was incubated with cross-linker in the same manner, higher order bands appeared at the earliest time point of 5 min (Fig. 3, middle right). The higher bands were consistent with molecular sizes of 2, 3, and 4 times the apparent molecular weight of n224 that was not treated with cross-linker at 0 min. Cross-linking results at both protein concentrations were similar, implying that the formation of n224 oligomers does not vary between 0.2 mg/ml (12 µM) and 1 mg/ml (61 µM).

Upon treatment of LZ HMW with EGS, shifted bands were observed at 5 min, with the monomer bands disappearing almost completely and three new bands of slower mobility appearing by 30 min (Fig. 3, bottom). LZ LMW cross-linking was identical. The new bands were too broad to accurately estimate molecular weights with respect to the standards, unlike the more distinct cross-linked bands seen on the full-length and n224 gels. The fuzzy bands of cross-linked LZ may be due to the mass of additional EGS moieties on the small LZ polypeptide and/or to additional cross-linking within the oligomer. It appears that the LZ polypeptide itself is sufficient to form higher order oligomers.

Analytical Ultracentrifugation—To further investigate the oligomeric state and overall shape of MSP3, analytical ultracentrifugation experiments were performed on the MSP3 polypeptides (Figs. 4, 5, and S2). For the full-length MSP3, the sedimentation velocity profiles showed two clearly separate boundaries, indicating the presence of two main species (Fig. 5) at ~4.7 and 5.8 S at a relative abundance of 71 and 29%, respectively, at a concentration of 10 µM. Both s values exceed the maximal theoretical values of compact hydrated spheres with the known molar mass and density of the monomer, from which it can be concluded that both species are oligomers.

In order to identify their nature further, sedimentation equilibrium experiments were conducted at three protein concentrations and three rotor speeds (Fig. 4A shows a single scan of this data). The average molar mass of the oligomers, determined with a global single species fit, was 126.5 kDa, with a root mean square (r.m.s.) deviation of 0.009 OD. Although this value is close to the trimer, one can rule this model out (except for representing the average molar mass), as we know from sedimentation velocity about the presence of two oligomers in significant abundance. Further, because the average molar mass is close to the trimer, we can rule out models with the smaller species equal or larger than the trimer. Therefore, we next modeled the sedimentation equilibrium data with a dimer-tetramer self-association model, resulting in a significantly better fit with an r.m.s. deviation of 0.005 OD at a KD of ~5 µM, which is qualitatively consistent with the relative population of species in sedimentation velocity. Other models, incorporating higher stoichiometries of the larger oligomer, resulted in significantly higher deviations (e.g. 0.015 OD for a dimer-hexamer model). Therefore, it can be concluded that the full-length protein is present in the form of dimers and tetramers. The fit with the dimer-tetramer model could be further refined, considering a low amount (11%) of incompetent dimer (r.m.s. deviation 0.0049 OD) (19), which led to a best fit KD value for the dimertetramer equilibrium of ~3 µM. In contrast, a model assuming an entirely stable mixture of noninteracting dimers and tetramers led to an r.m.s. deviation of 0.008 OD, a significant increase that supports the notion that the dimer and tetramer are in a reversible equilibrium.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4.
Sedimentation equilibrium analysis. Results for three of the six MSP3 polypeptides, FL, CC, and n224, are shown. A, absorbance profile of FL at 280 nm at a rotor speed of 12,000 rpm (circles) and best fit distribution from a dimer-tetramer self-association model (curve). B, absorbance profile of CC at 280 nm at a rotor speed of 25,000 rpm (circles) and the best fit distribution with a single, monomeric species of 18.8 kDa (curve). C, absorbance profile of n224 at 230 nm at a rotor speed of 20,000 rpm (circles) and theoretical distributions with a model (curve) for a single stable, tetrameric species of a best fit molecular mass of 69.4 kDa in the presence of trace impurities of 2.5 kDa. Due to the low extinction coefficient at 280 nm of the n224 construct, 230 nm was used in C. For clarity, every second data point is shown. The lower panels of A-C show the residuals of the fit of the model to the experimental data.

 
After identifying the oligomers present, it is possible to gain further information from the sedimentation velocity data. The presence of separate sedimentation boundaries and the similarity of boundary s values obtained over a range of concentrations (Fig. 5) clearly indicate a very slow association with a koff of less than 10-4/s (25), and the best fit value for koff from a global fit of the sedimentation profiles with a kinetically controlled dimer-tetramer model with SEDPHAT was <1 x 10-5/s, with KD values indicating approximately 1 order of magnitude weaker binding than from sedimentation equilibrium. Although this could be due to the different temperature (20 °C in velocity versus 4 °C in equilibrium sedimentation), it is notoriously difficult to determine equilibrium constants from very slow self-associating systems by sedimentation velocity due to the problem of establishing chemical equilibrium of species at the start of the sedimentation (32). Due to the extended time of sedimentation equilibrium experiments, we consider the sedimentation equilibrium KD more reliable and did not pursue this further.

Having established that the association is very slow, one can consider the s values of the sedimentation boundaries to represent the molecular hydrodynamic properties of the dimer and tetramer species. At rate constants of <1 x 10-5/s, the effects predicted by the Gilbert theory of chemical interconversion on the migration of the reaction boundary are negligible (25, 33). As a consequence, frictional ratios (f/f0) can be determined from the measured sedimentation coefficients. The values obtained were 1.64 for the dimer and 2.09 for the tetramer. For comparison, a smooth compact sphere would yield an f/f0 of 1.0. Since the f/f0 values are larger than 1.0, the results indicate that the full-length MSP3 dimer and tetramer are nonglobular and are highly extended in solution. Possible hydrodynamic models consistent with these f/f0 values are a prolate ellipsoid (cigar-shaped) and a oblate ellipsoid (discus-shaped). Using tabulated hydration data to estimate the contribution of hydration to the molecular frictional coefficients, the sedimentation velocity data can be used to make predictions about the ratio (a/b) of the ellipsoid major axis length, a, to its minor axis length, b. Assuming a prolate ellipsoid model, the MSP3 dimer would have an a/b ratio of 6.6 and dimensions of 250 x 37 Å. Assuming again a prolate ellipsoid model, the tetramer would have an a/b ratio of 14 and be ~500 Å by 37 Å. The difference in the frictional ratios for the dimer (f/f0 = 1.64) and tetramer (f/f0 = 2.09) is reflected in the doubling of the long axis of the hypothetical prolate ellipsoid and is consistent with an "end-to-end" association of the dimers to form the tetramer.

The dimensions of the equivalent prolate ellipsoid are dependent on the assumed degree of hydration. When using, instead of the value 0.56 g of H2O/g of protein (g/g) based on the amino acid composition and NMR-derived tabulated data (26), a value of 0.3 g/g supported by x-ray and neutron small angle scattering (34), prolate ellipsoid dimensions were calculated as 270 x 32 Å and 530 x 32 Å for the dimer and tetramer, respectively. Thus, although the absolute dimensions of the equivalent ellipsoid are dependent on the hydration value, we found that the approximate doubling of the long dimension was essentially invariant. This was true even when a hydration value of zero was assumed. These ultracentrifugation data confirm the size exclusion and chemical cross-linking findings that the full-length recombinant MSP3 forms dimers and tetramers with nonglobular, extended folds.

Sedimentation equilibrium data showed that the c308 and CC polypeptides are monomeric in solution, since the molecular mass determined by the equilibrium data is equal to that of the individual polypeptide (TABLE ONE). The sedimentation velocity results indicated that both proteins are nonglobular, with an f/f0 value of 1.9 for c308 and of 1.43 for CC. The large f/f0 value measured for c308 indicates that this monomeric form of MSP3 is highly elongated, with dimensions (assuming a prolate ellipsoid) of 250 x 24 Å, very similar to those of the full-length dimer. The elongated nature of both the dimeric full-length and monomeric c308 polypeptides suggests that oligomerization does not play a significant role in the observed asymmetry of the molecules and that it is inherent in the individual polypeptide itself. As for the CC polypeptide, its f/f0 value of 1.43 correlates well with the size exclusion results, since CC eluted at only a slightly smaller volume versus a standard protein, suggesting a moderately elongated shape (Fig. 2).


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Summary of sedimentation equilibrium and velocity results

 
The n224, LZ HMW, and LZ LMW polypeptides, which each contain the C-terminal domain shown by cross-linking to drive oligomer formation, were analyzed next. Sedimentation equilibrium results showed that each of these polypeptides formed stable oligomers, since the experimentally determined molecular mass of each species was larger than the prediction based on its sequence (TABLE ONE). The n224 and the LZ LMW were stable tetramers, but surprisingly, the molecular mass found for the LZ HMW species (45 kDa) is about 6 times that of the monomer (7.7 kDa) and thus predicts a hexamer. No dissociation of the hexamer was observed, and the data were not modeled well by a tetramer-octamer equilibrium (r.m.s. deviation of a global hexamer model was 0.005 OD, compared with 0.012 OD for the best fit tetramer-octamer model, globally fitting absorbance data acquired at multiple rotor speeds and wavelengths covering a concentration range from ~1 µM to 1.6 mM). It is evident that the isolated LZ domain is capable of forming stable higher order oligomers, probably of a coiled coil-like interaction.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 5.
Sedimentation coefficient distributions (s values) for the MSP3 polypeptides. Each curve represents a sedimentation velocity run for the labeled polypeptide. The y axis data of the CC distribution have been divided by a factor of 3 to fit on the plot. The s value distributions are corrected to standard conditions (20 °C, water as solvent). The dotted trace for the FL MSP3 shows the sedimentation coefficient distribution obtained at 8-fold lower concentration.

 
Sedimentation velocity results showed that the n224 and the LZ LMW tetramers are highly extended in shape, with f/f0 ratios close to 2.0 (TABLE ONE). Assuming an "end-to-end" interaction of prolate ellipsoid shaped dimers to form the tetramer, the n224 and LZ dimers would be about ~200 and ~120 Å long, respectively (half the length of the tetramer) (TABLE ONE). If all 55 residues of an LZ polypeptide formed a ideal {alpha}-helix with 3.6 residues and a 5.4-Å rise per turn and the 10 vector-derived residues adopted a random coil-like structure at the N terminus, the length of the LZ polypeptide would be close to the ellipsoidal LZ LMW dimer model length of 120 Å. This comparison allows us to predict that the LZ dimer is probably a side by side, or parallel, orientation of polypeptides and would hold true for the n224 and full-length dimers.

CD Measurements of the MSP3 Polypeptides—To investigate the type of secondary structure present in the MSP3 polypeptides, we examined them by far-UV CD spectroscopy (Fig. 6A). The CD data were deconvoluted using the programs CONTIN, CDSSTR, and SELCON3 (17), and the percentages of {alpha}-helix, {beta}-sheet, and random coil were estimated. The three algorithms were in good agreement with one another for all the determinations (±3%) except for the n224 data (discussed below).

The CD spectrum for full-length MSP3 in Fig. 6A shows the two minima around 208 and 222 nm that are characteristic of {alpha}-helices. Deconvolution of the data predicts ~47% {alpha}-helix, 6% {beta}-sheet, and 31% random coil for full-length MSP3, in good accordance with secondary structure predictions (see "Materials and Methods"). The estimated content of helix and random coil from CD is consistent with the sedimentation velocity results that predict an overall extended, elongated structure. The CD spectrum of the c308 protein predicts less {alpha}-helix (~33%) and only slightly more {beta}-sheet (~12%) and random coil (~36%), which is sensible, since c308 lacks the C-terminal leucine zipper-like helical domain. As such, the LZ LMW polypeptide spectrum (Fig. 6A) shows a strong {alpha}-helical signature and is estimated to be 31% helical and 29% random coil.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 6.
CD spectra of the MSP3 polypeptides. A, CD spectra of the full-length (FL), c308, CC, n224, and LZ (LMW) polypeptides recorded from 200 to 240 nm. B, temperature-induced unfolding of the full-length MSP3. The plot (inset) of mean residue ellipticity (m.r.e.) versus temperature represents the denaturation of the {alpha}-helices of the folded polypeptide.

 
The CD data estimate 45% {alpha}-helix, 8% {beta}-sheet, and 31% random coil for the CC protein. Fig. 6A shows that the spectrum of CC exhibits a different {alpha}-helical signature from that of the full-length protein. This may result from the helical segments of the coiled coil domain of CC being influenced less from neighboring residues than in the full-length protein (35).

For the n224 polypeptide, the CONTIN algorithm predicted 26% {beta}-sheet and 42% random coil, the SELCON3 algorithm predicted 9% {beta}-sheet and 22% random coil, and the CDSSTR algorithm was unable to make a prediction. The n224 spectrum in Fig. 6A reveals two minima moderately shifted from 208 and 222 nm, suggesting that n224 does contain some {alpha}-helix and a large amount of structure that is not represented in the sets of reference spectra on which standard deconvolution algorithms are based.

Thermostability of the Full-length MSP3 Protein—To assess the thermostability of the full-length protein, far-UV CD spectra of the protein were measured from 5 to 85 °C (Fig. 6B). It is clear from the spectra that the secondary structure denatures as the temperature is increased. Upon cooling, MSP3 refolded, since the 25 °C spectra before and after heating were similar (supplemental Fig. S3). When the mean residue ellipticity at 222 nm is plotted versus temperature (Fig. 6B, inset), the inflection point of the sigmoidal curve indicates a Tm of 50 ± 2 °C for the full-length protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our biophysical studies show that the recombinant full-length P. falciparum MSP3 possesses a highly extended and oligomeric structure that is 47% {alpha}-helix and 31% random coil. The full-length protein is in slow equilibrium between a dimeric and a tetrameric species, with a KD of tetramerization of 3 µM. The large f/f0 of the dimeric species, assuming an overall prolate ellipsoid shape and hydration of 0.56 g/g, suggests a molecule with dimensions of 250 x 37 Å (Fig. 7A). The tetrameric species has an even larger f/f0 that suggests an oligomeric protein of nearly 500 x 37 Å.

We have assumed a prolate ellipsoidal shape as a simple model for the gross asymmetry of the molecules. The detailed dimensions predicted for this model also depend on the hydration estimates. However, independent of any model shape and of the degree of hydration, the difference in the frictional ratios between the dimer and the tetramer shows that the two dimers making up the tetramer exhibit largely independent hydrodynamic drag forces. As a consequence of the independent drag forces, we conclude that the two dimers of a tetramer are not in close proximity and that tetramers do not have a reduced contour as compared with two separate dimers. Assuming this, our results suggest that the tetramer is a dimer of dimers that are arranged in an "end-to-end" orientation mediated through interactions at the C terminus (LZ) of the dimers (Fig. 7, B and C). This elongated structure explains the observed anomalous elution volumes of the full-length and shorter constructs on a size exclusion column (Fig. 2).

The biophysical data are consistent with the MSP3 dimer being formed via a parallel coiled coil interaction between the leucine zipper-like regions of two monomers. The dimers probably use their leucine zipper-like regions to make a four-helix coiled coil or bundle to form the MSP3 tetramer. The LZ region is necessary for tetramer formation, although the involvement of sequences that are N-terminal to LZ cannot be ruled out. The Glu-rich domain located N-terminal to the LZ region (Fig. 1, blue residues) is probably not involved due to poly-Glu peptides being largely unstructured and extended at neutral pH (36), as is consistent with the n224 CD spectra (Fig. 6A) and its broad peak on a size exclusion column (Fig. 2).

The importance of the N and C termini in the association of full-length MSP3 with the parasite surface has been reported (14, 37). In one experiment, washing of isolated merozoites resulted in the partitioning of a processed form of MSP3 lacking its N-terminal 54 residues into the soluble fraction, whereas the full-length protein remained associated with the merozoite (37). In another report, removal of the C-terminal 37 amino acid residues by gene knock-out resulted in loss of association of this truncated MSP3 polypeptide with the merozoite surface (14). We suggest that both termini of the protein are positioned at the same end of the molecule and near the membrane surface (Fig. 7A). Close proximity of the N terminus to the C-terminal oligomerization domain may explain why both dimers and tetramers are observed with the full-length protein, but only tetramers are observed with the n224 polypeptide that lacks the N-terminal 223 residues. The N-terminal regions appear to have a role in blocking or inhibiting dimers from forming tetramers.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7.
Models of P. falciparum MSP3. A, schematic representation of the dimeric full-length MSP3 protein based on our data and published reports. The stop/start points (black X) for the constructs and the relative position of proteolytic cleavage (orange X) observed in vivo (37) are shown. The domains are colored as in Fig. 1. B and C, two models of the MSP3 tetramer, both of which could lead to the asymmetric characteristics observed in the size exclusion chromatography (Fig. 2) and sedimentation velocity experiments (Fig. 5 and TABLE ONE). The Glu-rich regions are represented by thick blue lines. These drawings are not to scale, and additional secondary structure elements may be present.

 
The CC domain is a largely {alpha}-helical monomeric species in solution. The three Ala heptad repeat regions contained in the CC polypeptide (Fig. 1, red residues) have been proposed to form an intramolecular three-stranded coiled coil (10), and this would be consistent with our results. Alternatively, the repeat regions may form a two-stranded coiled coil structure (one example shown in Fig. 7A). The two-stranded model may be more accurate, since the CC polypeptide is moderately elongated (f/f0 = 1.43) with model prolate ellipsoid dimensions of 107 x 26 Å (TABLE ONE). That most of the sequence polymorphisms found in MSP3 map to the Ala heptad repeat region implies that it is exposed to immune system selection (3). A model of the MSP3 dimer that is consistent with our data and literature reports is shown in Fig. 7A. The dimer model would allow the coiled coil domains to interact with other proteins either on the parasite or red blood cell membranes.

The 31% random coil estimated from the CD experiments is consistent with many characteristics of MSP3 that also have also been observed in intrinsically unstructured proteins (38). MSP3 can be heated to 85 °C and cooled without precipitation. It has relatively few hydrophobic residues and is rich in charged amino acid residues (pI = 4.6) that make it migrate anomalously on SDS gels. Proteases readily cleave MSP3 and were controlled by our use of inhibitors, mutations, and His6 tags. The CD data for the n224 polypeptide suggest that much of the unstructured region may be contained in n224 itself, probably in the Glu-rich portion. Together with the helical regions, the unstructured portions help to form the highly extended MSP3 molecule.

Recently, it was noted that the MSP6, MSP11, and H101 proteins also possess a Glu-rich region located approximately the same distance from a C-terminal leucine zipper-like sequence and exhibit about 40-50% identity to the sequence of MSP3 (11, 13). We expect that these MSP3-like proteins can also form highly extended oligomers. The conservation of the Glu-rich region may allow these proteins a high degree of flexibility on the surface of the merozoite and/or allow them to extend their N termini far from the membrane surface. Electron microscopy studies on the blood stage merozoite have revealed thin fibrils or stalks of a proteinaceous nature that extend up to 300 Å from the membrane surface (39, 40). The elongated MSP3 oligomer and the likely elongated oligomers of the other family members are obvious candidates to be present in these structures. Since these proteins differ in amino acid sequence in their N termini regions, it is not difficult to imagine that these regions may direct interactions with various ligands on the red blood cell. Elucidating their function as long range receptors and/or scaffolding for other proteins requires further investigation.


   FOOTNOTES
 
* This work was supported by the Intramural Research Program of the NIAID, National Institutes of Health. 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

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed: Structural Biology Section, NIAID, Twinbrook II, 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-496-4773; Fax: 301-402-0284; E-mail: dgarboczi{at}niaid.nih.gov.

2 The abbreviations used are: MSP3, merozoite surface protein 3; DTT, dithiothreitol; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; NTA, nitrilotriacetic acid; EGS, ethylene glycol bis-(succinimidylsuccinate); Bis-Tris, bis(2-hydroxyethyl)imminotris(hydroxymethyl)methane; HMW, high molecular weight; LMW, low molecular weight; r.m.s., root mean square. Back


   ACKNOWLEDGMENTS
 
We thank Mark Garfield of the Research Technologies Branch, NIAID, National Institutes of Health, for help with the N-terminal sequencing and mass spectrometry analysis of the MSP3 proteins. We also thank Scott Garman and the other members of the Structural Biology Section for helpful comments and insights on this work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y., and Hay, S. I. (2005) Nature 434, 214-217[CrossRef][Medline] [Order article via Infotrieve]
  2. McColl, D. J., Silva, A., Foley, M., Kun, J. F., Favaloro, J. M., Thompson, J. K., Marshall, V. M., Coppel, R. L., Kemp, D. J., and Anders, R. F. (1994) Mol. Biochem. Parasitol. 68, 53-67[CrossRef][Medline] [Order article via Infotrieve]
  3. McColl, D. J., and Anders, R. F. (1997) Mol. Biochem. Parasitol. 90, 21-31[CrossRef][Medline] [Order article via Infotrieve]
  4. Hisaeda, H., Saul, A., Reece, J. J., Kennedy, M. C., Long, C. A., Miller, L. H., and Stowers, A. W. (2002) J. Infect. Dis. 185, 657-664[CrossRef][Medline] [Order article via Infotrieve]
  5. Carvalho, L. J., Oliveira, S. G., Theisen, M., Alves, F. A., Andrade, M. C., Zanini, G. M., Brigido, M. C., Oeuvray, C., Povoa, M. M., Muniz, J. A., Druilhe, P., and Daniel-Ribeiro, C. T. (2004) Scand. J. Immunol. 59, 363-372[CrossRef][Medline] [Order article via Infotrieve]
  6. Oeuvray, C., Bouharoun-Tayoun, H., Gras-Masse, H., Bottius, E., Kaidoh, T., Aikawa, M., Filgueira, M. C., Tartar, A., and Druilhe, P. (1994) Blood 84, 1594-1602[Abstract/Free Full Text]
  7. Galinski, M. R., Corredor-Medina, C., Povoa, M., Crosby, J., Ingravallo, P., and Barnwell, J. W. (1999) Mol. Biochem. Parasitol. 101, 131-147[CrossRef][Medline] [Order article via Infotrieve]
  8. Okenu, D. M., Thomas, A. W., and Conway, D. J. (2000) Mol. Biochem. Parasitol. 109, 185-188[Medline] [Order article via Infotrieve]
  9. Hudson, D. E., Miller, L. H., Richards, R. L., David, P. H., Alving, C. R., and Gitler, C. (1983) J. Immunol. 130, 2886-2890[Abstract]
  10. Mulhern, T. D., Howlett, G. J., Reid, G. E., Simpson, R. J., McColl, D. J., Anders, R. F., and Norton, R. S. (1995) Biochemistry 34, 3479-3491[Medline] [Order article via Infotrieve]
  11. Trucco, C., Fernandez-Reyes, D., Howell, S., Stafford, W. H., Scott-Finnigan, T. J., Grainger, M., Ogun, S. A., Taylor, W. R., and Holder, A. A. (2001) Mol. Biochem. Parasitol. 112, 91-101[CrossRef][Medline] [Order article via Infotrieve]
  12. Pearce, J. A., Triglia, T., Hodder, A. N., Jackson, D. C., Cowman, A. F., and Anders, R. F. (2004) Infect. Immun. 72, 2321-2328[Abstract/Free Full Text]
  13. Pearce, J. A., Mills, K., Triglia, T., Cowman, A. F., and Anders, R. F. (2005) Mol. Biochem. Parasitol. 139, 141-151[CrossRef][Medline] [Order article via Infotrieve]
  14. Mills, K. E., Pearce, J. A., Crabb, B. S., and Cowman, A. F. (2002) Mol. Microbiol. 43, 1401-1411[CrossRef][Medline] [Order article via Infotrieve]
  15. Sodeoka, M., Larson, C. J., Chen, L., LeClair, K. P., and Verdine, G. L. (1993) Bioorg. Med. Chem. Lett. 3, 1089-1094
  16. Chen, J., Wharton, S. A., Weissenhorn, W., Calder, L. J., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12205-12209[Abstract/Free Full Text]
  17. Sreerama, N., and Woody, R. W. (2000) Anal. Biochem. 287, 252-260[CrossRef][Medline] [Order article via Infotrieve]
  18. Schuck, P., and Braswell, E. H. (2000) in Current Protocols in Immunology (Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., ed) pp. 18.8.1-18.8.22, John Wiley & Sons, Inc., New York
  19. Vistica, J., Dam, J., Balbo, A., Yikilmaz, E., Mariuzza, R. A., Rouault, T. A., and Schuck, P. (2004) Anal. Biochem. 326, 234-256[CrossRef][Medline] [Order article via Infotrieve]
  20. Balbo, A., and Schuck, P. (2005) in Protein-Protein Interactions (Golemis, E., and Adams, P. D., eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, in press
  21. Svedberg, T., and Pederson, K. O. (1940) The Ultracentrifuge, Oxford University Press, London
  22. Schuck, P. (2000) Biophys. J. 78, 1606-1619[Medline] [Order article via Infotrieve]
  23. Schuck, P., Perugini, M. A., Gonzales, N. R., Howlett, G. J., and Schubert, D. (2002) Biophys. J. 82, 1096-1111[Medline] [Order article via Infotrieve]
  24. Schuck, P., and Demeler, B. (1999) Biophys. J. 76, 2288-2296[Medline] [Order article via Infotrieve]
  25. Dam, J., Velikovsky, C. A., Mariuzza, R. A., Urbanke, C., and Schuck, P. (2005) Biophys. J. 89, 619-634[CrossRef][Medline] [Order article via Infotrieve]
  26. Kuntz, I. D. (1971) J. Am. Chem. Soc. 93, 514-516[CrossRef]
  27. Van Holde, K. E., Johnson, W. C., and Ho, P. S. (2005) Principles of Physical Biochemistry, 2nd Ed., pp. 223-248, Prentice-Hall, Upper Saddle River, NJ
  28. Lebowitz, J., Lewis, M. S., and Schuck, P. (2002) Protein Sci. 11, 2067-2079[CrossRef][Medline] [Order article via Infotrieve]
  29. Geourjon, C., and Deleage, G. (1995) Comput. Appl. Biosci. 11, 681-684[Abstract/Free Full Text]
  30. King, R. D., Saqi, M., Sayle, R., and Sternberg, M. J. (1997) Comput. Appl. Biosci. 13, 473-474[Free Full Text]
  31. Frishman, D., and Argos, P. (1997) Proteins 27, 329-335[CrossRef][Medline] [Order article via Infotrieve]
  32. Yikilmaz, E., Rouault, T. A., and Schuck, P. (2005) Biochemistry 44, 8470-8478[CrossRef][Medline] [Order article via Infotrieve]
  33. Gilbert, G. A. (1959) Proc. R. Soc. Lond. A 250, 377-388[Abstract/Free Full Text]
  34. Perkins, S. J. (2001) Biophys. Chem. 93, 129-139[CrossRef][Medline] [Order article via Infotrieve]
  35. Yang, J. T., Wu, C. S., and Martinez, H. M. (1986) Methods Enzymol. 130, 208-269[Medline] [Order article via Infotrieve]
  36. Kimura, T., Takahashi, S., Akiyama, S., Uzawa, T., Ishimori, K., and Morishima, I. (2002) J. Am. Chem. Soc. 124, 11596-11597[CrossRef][Medline] [Order article via Infotrieve]
  37. Pearce, J. A., Hodder, A. N., and Anders, R. F. (2004) Exp. Parasitol. 108, 186-189[Medline] [Order article via Infotrieve]
  38. Tompa, P. (2002) Trends Biochem. Sci. 27, 527-533[CrossRef][Medline] [Order article via Infotrieve]
  39. Galinski, M. R., and Barnwell, J. W. (1996) Parasitol. Today 12, 20-29[CrossRef][Medline] [Order article via Infotrieve]
  40. Bannister, L. H., Mitchell, G. H., Butcher, G. A., Dennis, E. D., and Cohen, S. (1986) Cell Tissue Res. 245, 281-290[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
 Am J Trop Med HygHome page
S. J. Jordan, O. H. Branch, J. C. Castro, R. A. Oster, and J. C. Rayner
Genetic Diversity of the Malaria Vaccine Candidate Plasmodium falciparum Merozoite Surface Protein-3 in a Hypoendemic Transmission Environment
Am J Trop Med Hyg, March 1, 2009; 80(3): 479 - 486.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. W. Kauth, U. Woehlbier, M. Kern, Z. Mekonnen, R. Lutz, N. Mucke, J. Langowski, and H. Bujard
Interactions between Merozoite Surface Proteins 1, 6, and 7 of the Malaria Parasite Plasmodium falciparum
J. Biol. Chem., October 20, 2006; 281(42): 31517 - 31527.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
R. Lundquist, L. K. Nielsen, A. Jafarshad, D. SoeSoe, L. H. Christensen, P. Druilhe, and M. H. Dziegiel
Human Recombinant Antibodies against Plasmodium falciparum Merozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties.
Infect. Immun., June 1, 2006; 74(6): 3222 - 3231.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/44/37236    most recent
M506753200v1
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 Burgess, B. R.
Right arrow Articles by Garboczi, D. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burgess, B. R.
Right arrow Articles by Garboczi, D. N.
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 © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement