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Results for “Solvent accessibility”

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PubMed2006

Coupling hydrophobicity, dispersion, and electrostatics in continuum solvent models.

An implicit solvent model is presented that couples hydrophobic, dispersion, and electrostatic solvation energies by minimizing the system Gibbs free energy with respect to the solvent volume exclusion function. The solvent accessible surface is the output of the theory. The method is illustrated with the solvation of simple solutes on different length scales and captures the sensitivity of hydration to the particular form of the solute-solvent interactions in agreement with recent computer simulations.

Alkanes↗

PubMed1987

Inaccessibility of tryptophan residues of recombinant human renin to quenching agents.

The fluorescence quenching of the three tryptophan residues of recombinant human renin was determined using ionic and penetrating quenchers. Tryptophans 44,200, and 312 of recombinant human renin were found to be totally inaccessible to the ionic quenchers cesium and iodide and only partially accessible to the penetrating quencher acrylamide. The renin had a fluorescence emission maximum at 325 nm which was made up of three separate components as determined by second derivative spectroscopy. These data are in accord with solvent accessibility calculations from three-dimensional models of human renin but differ from findings published previously from similar analysis of mouse submandibular gland renin (Quay, S. C., Heropoulous, A., Commes, K., Dzau, V. J. (1985) J. Biol. Chem. 260, 15055-15058), which is 68% identical in sequence to human renin.

Acrylamide↗

PubMed2002

The transmembrane domains of the ABC multidrug transporter LmrA form a cytoplasmic exposed, aqueous chamber within the membrane.

The ABC multidrug transporter LmrA of Lactococcus lactis consists of six putative transmembrane segments (TMS) and a nucleotide binding domain. LmrA functions as a homodimer in which the two membrane domains form the solute translocation path across the membrane. To obtain structural information of LmrA a cysteine scanning accessibility approach was used. Cysteines were introduced in the cysteine-less wild-type LmrA in each hydrophilic loop and in TMS 6, and each membrane-embedded aromatic residue was mutated to cysteine. Of the 41 constructed single cysteine mutants, only one mutant, L301C, was not expressed. Most single-cysteine mutants were capable of drug transport and only three mutants, F37C, M299C, and N300C, were inactive, indicating that none of the aromatic residues in the transmembrane regions of LmrA are crucial for substrate binding or transport. Modification of the active mutants with N-ethylmaleimide blocked the transport activity in five mutants (S132C, L174C, S206C, S234C, and L292C). All cysteine residues in external and internal loops were accessible to fluorescein maleimide. The labeling experiments also showed that this thiol reagent cannot cross the membrane under the conditions used and confirmed the presence of six TMSs in each monomeric half of the transporter. Surprisingly, several single cysteines in the predicted TMSs could also be labeled by the bulky fluorescein maleimide molecule, suggesting unrestricted accessibility via an aqueous pathway. The periodicity of fluorescein maleimide accessibility of residues 291 to 308 in TMS 6 showed that this membrane-spanning alpha-helix has one face of the helix exposed to an aqueous cavity along its full-length. This finding, together with the solvent accessibility of 11 of 15 membrane-embedded aromatic residues, indicates that the transmembrane domains of the LmrA transporter form, under nonenergized conditions, an aqueous chamber within the membrane, which is open to the intracellular milieu.

Amino Acid Sequence↗

PubMed2003

Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway.

This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [3H]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed </=25% Gly-Sar uptake when compared with WT-hPepT1. P182C-hPepT1 showed approximately 40% specific activity whereas all the remaining transporters, although still sensitive to single cysteine mutations, exhibited more than 50% specific activity when compared to WT-hPepT1. The activity of F166C-, L176C-, S177C-, T178C-, I180C-, T181C-, and P182C-hPepT1 was partially inhibited, while the activity of F163C- and I170C-hPepT1 was completely inhibited by 2.5mM MTSEA. F163C, I165C, F166C, A169C, I170C, S177C, T181C, and P182C were clearly accessible to 1mM MTSET. Overall, these results suggest that TMS5 lines the putative aqueous channel and is slightly tilted from the vertical axis of the channel, with the exofacial half forming a classical amphipathic alpha-helix and the cytoplasmic half being highly solvent accessible.

Animals↗

PubMed2004

Solvation forces on biomolecular structures: a comparison of explicit solvent and Poisson-Boltzmann models.

Continuum electrostatics methods have become increasingly popular due to their ability to provide approximate descriptions of solvation energies and forces without expensive sampling required by explicit solvent models. In particular, the Poisson-Boltzmann equation (PBE) provides electrostatic potentials, solvation energies, and forces by modeling the solvent as a featureless, dielectric material, and the mobile ions as a continuous distribution of charge. Polar solvation forces and energies obtained from the PBE are often supplemented with simple solvent-accessible surface area (SASA) models of nonpolar solvation. Given the recent development of methods that enable the use of PBE and SASA forces in molecular dynamics simulations, it is important to determine the ability of these implicit solvent models to accurately reproduce the solvation forces of more detailed explicit solvent simulations. In this article, we compare PBE and SASA solvation forces with explicit solvent forces for several snapshots from eight trajectories of static conformations of intestinal fatty acid binding protein. The results from this comparison show that current implementations of the PBE are capable of generating polar solvation forces that correlate well with explicit solvent forces but systematically overestimate the magnitude of the interaction. However, SASA-based nonpolar forces are found to have no significant correlation with nonpolar explicit solvent forces. Nevertheless, due to the small magnitude of the nonpolar forces in the current system, a good correlation is still obtained for total solvation forces. The good correlation of implicit solvent forces with more detailed explicit solvent models is encouraging and implies that the systematic errors identified in these models could be corrected by appropriate parameterization of the force fields.

Computer Simulation↗

PubMed1986

Intrinsic fluorescence studies on saccharide binding to Artocarpus integrifolia lectin.

The combining region of Artocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-1, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The alpha-anomer of galactose binds more strongly than its beta-counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin. The lectin binds to the Thomsen-Friedenreich antigen (Gal beta(1----3)GalNAc) more strongly than the other disaccharides studied, viz. Gal beta (1----4) Gal and Gal beta (1----3) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region of Artocarpus lectin is complementary to that of T-antigen. Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.

Disaccharides↗

PubMed1991

Mapping electrostatic interactions in macromolecular associations.

In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed. An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers. Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive. We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation. The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces. Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains. We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex. Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins. Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein. Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal. This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.

Amino Acids↗

PubMed1998

Identification of a beta-lactoglobulin lactosylation site.

Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that beta-lactoglobulin (beta-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of beta-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of beta-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.

Amino Acid Sequence↗

PubMed1988

Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase.

The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed2001

Comparison between light induced and chemically induced oxidation of rhVEGF.

PURPOSE: The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. METHODS: A liquid formulation of rhVEGF was exposed to fluorescent light (2 x 10(4) lux for up to 4 weeks), hydrogen peroxide (H2O2), or t-butythydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. RESULTS: Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78.81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%. possibly due to the role of Met 18in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H2O2, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H2O2 and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. CONCLUSIONS: Methionine residues with exposed surface areas greater than 65 A2 and sulfur surface areas greater than 16 A2 were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H2O2 to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 A2, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.

Amino Acid Sequence↗

PubMed1989

Preparation, characterization and application of interleukin-1 beta mutant proteins with surface-accessible cysteine residues.

Two mutants of interleukin-1 beta (K27C and K138C) were produced using site-specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 1H-NMR spectroscopy and shown to be similar to the wild-type protein. The receptor-binding affinity and biological activity of K27C and K138C were also similar to wild-type protein. The substituted cysteines in both mutant proteins were shown to be solvent-accessible as judged by their reactivity towards sulfhydryl reagents. As the wild-type protein contains two cysteines, which are both solvent-inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequence-specific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide-linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor-binding affinities to that of the wild-type unconjugated protein and were found suitable for flow-cytometric analysis.

Alkylation↗

PubMed1997

A 3D-1D substitution matrix for protein fold recognition that includes predicted secondary structure of the sequence.

In protein fold recognition, a probe amino acid sequence is compared to a library of representative folds of known structure to identify a structural homolog. In cases where the probe and its homolog have clear sequence similarity, traditional residue substitution matrices have been used to predict the structural similarity. In cases where the probe is sequentially distant from its homolog, we have developed a (7 x 3 x 2 x 7 x 3) 3D-1D substitution matrix (called H3P2), calculated from a database of 119 structural pairs. Members of each pair share a similar fold, but have sequence identity less than 30%. Each probe sequence position is defined by one of seven residue classes and three secondary structure classes. Each homologous fold position is defined by one of seven residue classes, three secondary structure classes, and two burial classes. Thus the matrix is five-dimensional and contains 7 x 3 x 2 x 7 x 3 = 882 elements or 3D-1D scores. The first step in assigning a probe sequence to its homologous fold is the prediction of the three-state (helix, strand, coil) secondary structure of the probe; here we use the profile based neural network prediction of secondary structure (PHD) program. Then a dynamic programming algorithm uses the H3P2 matrix to align the probe sequence with structures in a representative fold library. To test the effectiveness of the H3P2 matrix a challenging, fold class diverse, and cross-validated benchmark assessment is used to compare the H3P2 matrix to the GONNET, PAM250, BLOSUM62 and a secondary structure only substitution matrix. For distantly related sequences the H3P2 matrix detects more homologous structures at higher reliabilities than do these other substitution matrices, based on sensitivity versus specificity plots (or SENS-SPEC plots). The added efficacy of the H3P2 matrix arises from its information on the statistical preferences for various sequence-structure environment combinations from very distantly related proteins. It introduces the predicted secondary structure information from a sequence into fold recognition in a statistical way that normalizes the inherent correlations between residue type, secondary structure and solvent accessibility.

Algorithms↗

PubMed1998

Protein hydration and unfolding--insights from experimental partial specific volumes and unfolded protein models.

BACKGROUND: The partial specific volume of a protein is an experimental quantity containing information about solute-solvent interactions and protein hydration. We use a hydration-shell model to partition the partial specific volume into an intrinsic volume occupied by the protein and a change in the volume occupied by the solvent resulting from the solvent interactions with the protein. We seek to extract microscopic information about protein hydration and unfolding from experimental volume measurements without using computer simulations. We employ the idea that the protein-solvent interaction will be proportional to the surface area of the protein. RESULTS: A linear relationship is obtained when the difference between the experimental protein partial specific volume and its intrinsic volume is plotted as a function of the protein solvent-accessible surface area. The effect of using different protein volume definitions on the analysis of protein volumetric properties is discussed. Volumetric data are used to test a model for the unfolded state of proteins and to make predictions about the denatured state. CONCLUSIONS: The linear relationship between hydration-shell volume change and accessible surface area reflects the similar surface properties (fractional composition of nonpolar, polar and charged surface) among a diverse set of proteins. This linear relationship is found to be independent of how the solution is partitioned into solute and solvent components. The interpretation of hydration shell versus bulk water properties is found to be very model dependent, however. The maximally exposed unfolded protein model is found to be inconsistent with experimental volume changes of unfolding.

Algorithms↗

PubMed2006

Characterization of the hydrodynamic properties of the folding transition state of an SH3 domain by magnetization transfer NMR spectroscopy.

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than approximately 1 M) and glycerol enables determination of alpha values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 +/- 360 A(2)) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.

Animals↗

PubMed2003

Direct probing of RNA structures and RNA-protein interactions in the HIV-1 packaging signal by chemical modification and electrospray ionization fourier transform mass spectrometry.

RNA hairpins of the HIV-1 packaging signal and their complexes with the nucleocapsid protein p7 (NC) were probed by solvent-accessibility reagents and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The combination of dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMCT) offers the full range of information on base-pairing and solvent exposure concerning the four more abundant ribonucleotides. ESI-FTMS provides a universal method to achieve a direct and unambiguous characterization of alkylated structures, with no need for the different probe-specific procedures required by established methodologies based on gel electrophoresis. It enables us to streamline the optimization of the conditions for probe administration to minimize the incidence of probe-induced distortion of the structures under investigation. Nucleotides located in the single-stranded loops of hairpins SL2, SL3 and SL4 manifested different levels of protection, which were correlated directly to their conformation and structural surroundings. A common feature noted for all the hairpins was the limited susceptibility observed for the guanine base located at the 5'-end of each tetraloop, which assumes a stacked position upon the last base-pair of the double-stranded stems. The remaining loop bases were found to be clearly accessible by modifying reagents in free RNA, but were effectively protected in the NC-hairpin complexes. While this finding is consistent with the proven participation of SL2 and SL3 loops in interactions with NC, it contrasts with prior suggestions that tetraloop bases in SL4 might not be involved directly in NC binding. Alkylation was detected for stem nucleotides, which are not involved in the normal base-pairing and stacking typical of double-stranded structures, such as adenine 15 of the SL2 triple-base platform. Modification of the blunt ends of the double-stranded stems was found to be absent or extremely limited, due to the annealing stabilization introduced by the presence of G-C pairs at the end of the stems structures. Previously undetected alkylation of guanine 3 and guanine 13 in SL4 provides direct evidence of the destabilizing effects induced by the tandem G.U wobbles on the double-stranded structure of this stem, which is thought to be important for the hairpin's biological function.

Base Sequence↗

PubMed2002

Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.

We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional when co-reconstituted with the affinity-purified Ca-ATPase in liposomes. ANSmal emission properties and its solvent accessibility indicate that Cys-24 is in an aqueous environment outside the membrane. Fluorescence quenching and time-resolved anisotropy measurements of ANSmal-PLB demonstrate distinct structures for PLB in the free and Ca-ATPase-bound state. Both solvent exposure and probe motions of ANSmal are enhanced upon interaction of PLB with the Ca-ATPase. This conformational transition entails conversion of free PLB in a conformation which is insensitive to one which is sensitive to the phosphorylation state of PLB. Upon phosphorylation of Ca-ATPase-bound PLB, a decreased level of solvent exposure of ANSmal is observed, suggesting that the amino acid sequence of PLB near the lipid-water interface acts as a conformational switch in response to the phosphorylation of PLB. A longer correlation time, resolved by anisotropy measurements, corresponding to polypeptide chain fluctuations, is substantially restricted by interaction of PLB with the Ca-ATPase. This restriction is not reversed by phosphorylation of PLB, indicating that the region around Cys-24 near the lipid-water interface does not undergo dissociation from the Ca-ATPase. These results suggest that the phosphorylation by PKA induces a redistribution of PLB-Ca-ATPase protein contacts to relieve the inhibitory effect of PLB for the activation of calcium transport.

Acrylamide↗

PubMed2005

Flexibility of metal binding sites in proteins on a database scale.

Protein metal binding sites in the pre-bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo-holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side-chain rearrangement inversely correlates with the number of first-shell residues. Analysis of side-chain reorientations as a result of metal binding showed that in 95% of the rigid-backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre-bound form. The frequencies of side-chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state.

Apoproteins↗

PubMed1991

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4.

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.

Amino Acid Sequence↗