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PubMed2005

Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein l30e.

The origin of reduced heat capacity change of unfolding (DeltaC(p)) commonly observed in thermophilic proteins is controversial. The established theory that DeltaC(p) is correlated with change of solvent-accessible surface area cannot account for the large differences in DeltaC(p) observed for thermophilic and mesophilic homologous proteins, which are very similar in structures. We have determined the protein stability curves, which describe the temperature dependency of the free energy change of unfolding, for a thermophilic ribosomal protein L30e from Thermococcus celer, and its mesophilic homologue from yeast. Values of DeltaC(p), obtained by fitting the free energy change of unfolding to the Gibbs-Helmholtz equation, were 5.3 kJ mol(-1) K(-1) and 10.5 kJ mol(-1) K(-1) for T.celer and yeast L30e, respectively. We have created six charge-to-neutral mutants of T.celer L30e. Removal of charges at Glu6, Lys9, and Arg92 decreased the melting temperatures of T.celer L30e by approximately 3-9 degrees C, and the differences in melting temperatures were smaller with increasing concentration of salt. These results suggest that these mutations destabilize T.celer L30e by disrupting favorable electrostatic interactions. To determine whether electrostatic interactions contribute to the reduced DeltaC(p) of the thermophilic protein, we have determined DeltaC(p) for wild-type and mutant T.celer L30e by Gibbs-Helmholtz and by van't Hoff analyses. A concomitant increase in DeltaC(p) was observed for those charge-to-neutral mutants that destabilize T.celer L30e by removing favorable electrostatic interactions. The crystal structures of K9A, E90A, and R92A, were determined, and no structural change was observed. Taken together, our results support the conclusion that electrostatic interactions contribute to the reduced DeltaC(p) of T.celer L30e.

Archaeal Proteins↗

PubMed2003

Solvation parameters for predicting the structure of surface loops in proteins: transferability and entropic effects.

A new procedure for optimizing parameters of implicit solvation models introduced by us has been applied successfully first to cyclic peptides and more recently to three surface loops of ribonuclease A (Das and Meirovitch, Proteins 2001;43:303-314) using the simplified model E(tot) = E(FF)(epsilon = nr) + Sigma(i) sigma(i)A(i), where sigma(i) are atomic solvation parameters (ASPs) to be optimized, A(i) is the solvent accessible surface area of atom i, E(FF)(epsilon = nr) is the AMBER force-field energy of the loop-loop and loop-template interactions with a distance-dependent dielectric constant, epsilon = nr, where n is a parameter. The loop is free to move while the protein template is held fixed in its X-ray structure; an extensive conformational search for energy minimized loop structures is carried out with our local torsional deformation method. The optimal ASPs and n are those for which the structure with the lowest minimized energy [E(tot)(n,sigma(i))] becomes the experimental X-ray structure, or less strictly, the energy gap between these structures is within 2-3 kcal/mol. To check if a set of ASPs can be defined, which is transferable to a large number of loops, we optimize individual sets of ASPs (based on n = 2) for 12 surface loops from which an "averaged" best-fit set is defined. This set is then applied to the 12 loops and an independent "test" group of 8 loops leading in most cases to very small RMSD values; thus, this set can be useful for structure prediction of loops in homology modeling. For three loops we also calculate the free energy gaps to find that they are only slightly smaller than their energy counterparts, indicating that only larger n will enable reducing too large gaps. Because of its simplicity, this model allowed carrying out an extensive application of our methodology, providing thereby a large number of benchmark results for comparison with future calculations based on n > 2 as well as on more sophisticated solvation models with as yet unknown performance for loops.

Amino Acid Sequence↗

PubMed1997

Structures of N-termini of helices in proteins.

We have surveyed 393 N-termini of alpha-helices and 156 N-termini of 3(10)-helices in 85 high resolution, non-homologous protein crystal structures for N-cap side-chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N-cap sites. The following rules are generally observed for N-capping in alpha-helices: Thr and Ser N-cap side chains adopt the gauche - rotamer, hydrogen bond to the N3 NH and have psi restricted to 164 +/- 8 degrees. Asp and Asn N-cap side chains either adopt the gauche - rotamer and hydrogen bond to the N3 NH with psi = 172 +/- 10 degrees, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with psi = 1-7 +/- 19 degrees. With all other N-caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N-terminus by blocking solvation and psi is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N-cap backbone C = O in more likely to form at the N-terminus when an unfavorable N-cap is present. In the 3(10)-helix Asn and Asp remain favorable N-caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N-cap when alpha-helix formation is to be encouraged while 3(10)-helix formation discouraged. The strong energetic and structural preferences found for N-caps, which differ greatly from positions within helix interiors, suggest that N-caps should be treated explicitly in any consideration of helical structure in peptides or proteins.

Hydrogen Bonding↗

PubMed2001

Implicit solvation in the self-consistent mean field theory method: sidechain modelling and prediction of folding free energies of protein mutants.

The Atomic Solvation Parameter (ASP) model is one of the simplest models of solvation, in which the solvation free energy of a molecule is proportional to the solvent accessible surface area (SAS) of its atoms. However, until now this model had not been incorporated into the Self-Consistent Mean Field Theory (SCMFT) method for modelling sidechain conformations in proteins. The reason for this is that SAS is a many-body quantity and, thus, it is not obvious how to define it within the Mean Field (MF) framework, where multiple copies of each sidechain exist simultaneously. Here, we present a method for incorporating an SAS-based potential, such as the ASP model, into SCMFT. The theory on which the method is based is exact within the MF framework, that is, it does not depend on a pairwise or any other approximation of SAS. Therefore, SAS can be calculated to arbitrary accuracy. The method is computationally very efficient: only 7.6% slower on average than the method without solvation. We applied the method to the prediction of sidechain conformation, using as a test set high-quality solution structures of 11 proteins. Solvation was found to substantially improve the prediction accuracy of well-defined surface sidechains. We also investigated whether the methodology can be applied to prediction of folding free energies of protein mutants, using a set of barnase mutants. For apolar mutants, the modest correlation observed between calculated and observed folding free energies without solvation improved substantially when solvation was included, allowing the prediction of trends in the folding free energies of this type of mutants. For polar mutants, correlation was not significant even with solvation. Several other factors also responsible for the correlation were identified and analysed. From this analysis, future directions for applying and improving the present methodology are discussed.

Algorithms↗

PubMed1992

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.

Amino Acids↗

PubMed1996

The enthalpy change in protein folding and binding: refinement of parameters for structure-based calculations.

Two effects are mainly responsible for the observed enthalpy change in protein unfolding: the disruption of internal interactions within the protein molecule (van der Waals, hydrogen bonds, etc.) and the hydration of the groups that are buried in the native state and become exposed to the solvent on unfolding. In the traditional thermodynamic analysis, the effects of hydration have usually been evaluated using the thermodynamic data for the transfer of small model compounds from the gas phase to water. The contribution of internal interactions, on the other hand, are usually estimated by subtracting the hydration effects from the experimental enthalpy of unfolding. The main drawback of this approach is that the enthalpic contributions of hydration, and those due to the disruption of internal interactions, are more than one order of magnitude larger than the experimental enthalpy value. The enthalpy contributions of hydration and disruption of internal interactions have opposite signs and cancel each other almost completely resulting in a final value that is over 10 times smaller than the individual terms. For this reason, the classical approach cannot be used to accurately predict unfolding enthalpies from structure: any error in the estimation of the hydration enthalpy will be amplified by a factor of 10 or more in the estimation of the unfolding enthalpy. Recently, it has been shown that simple parametric equations that relate the enthalpy change with certain structural parameters, especially changes in solvent accessible surface areas have considerable predictive power. In this paper, we provide a physical foundation to that parametrization and in the process we present a system of equations that explicitly includes the enthalpic effects of the packing density between the different atoms within the protein molecule. Using this approach, the error in the prediction of folding/unfolding enthalpies at 60 degrees C, the median temperature for thermal unfolding, is better than +/- 3% (standard deviation = 4 kcal/mol).

Protein Conformation↗

PubMed1996

A method for detecting hydrophobic patches on protein surfaces.

A method for the detection of hydrophobic patches on the surfaces of protein tertiary structures is presented. It delineates explicit contiguous pieces of surface of arbitrary size and shape that consist solely of carbon and sulphur atoms using a dot representation of the solvent-accessible surface. The technique is also useful in detecting surface segments with other characteristics, such as polar patches. Its potential as a tool in the study of protein-protein interactions and substrate recognition is demonstrated by applying the method to myoglobin, Leu/IIe/Val-binding protein, lipase, lysozyme, azurin, triose phosphate isomerase, carbonic anhydrase, and phosphoglycerate kinase. Only the largest patches, having sizes exceeding random expectation, are deemed meaningful. In addition to well-known hydrophobic patches on these proteins, a number of other patches are found, and their significance is discussed. The method is simple, fast, and robust. The program text is obtainable by anonymous ftp.

Models, Molecular↗

PubMed1997

Better 1D predictions by experts with machines.

Accuracy of predicting protein secondary structure and solvent accessibility has been improved significantly by using evolutionary information contained in multiple sequence alignments. For the second Asilomar meeting, predictions were made automatically for all targets using the publicly available prediction service PredictProtein. Additionally, a semiautomatic procedure for generating more informative alignments was used in combination with the PHD prediction methods. Results confirmed the estimates for prediction accuracy. Furthermore, the more informative alignments yielded better predictions. The fairly accurate predictions of 1D structure were successfully used by various groups for the Asilomar meeting as first step toward predicting higher dimensions of protein structure.

Expert Systems↗

PubMed1999

Exploring protein interiors: the role of a buried histidine in the KH module fold.

The K-homology (KH) module is a novel RNA-binding motif. The structures of a representative KH motif from vigilin (vig-KH6) and of the first KH domain of fmr1 have been recently solved by nuclear magnetic resonance (NMR) and automated assignment-refinement techniques (ARIA). While a hydrophobic residue is found at position 21 in most of the KH modules, a buried His is conserved in all the 15 KH repeats of vigilin. This position must therefore have a key structural role in stabilizing the hydrophobic core. In the present work, we have addressed the following questions in order to obtain a detailed description of the role of His 21: i) what is the exact role of the histidine in the hydrophobic core of vig-KH6? ii) can we define the interactions that allow a conserved buried position to be occupied by a histidine both in vig-KH6 and in the whole vigilin KH sub-family? iii) how is the structure and stability of vig-KH6 influenced by the state of protonation of this histidine? To answer these questions, we have carried out an extensive refinement of the vig-KH6 structure using both an improved ARIA protocol starting from different initial structures and successively running restrained and unrestrained trajectories in water. An analysis of the stability of secondary structural elements, solvent accessibility, and hydrogen bonding patterns allows hypothesis on the structural role of residue His 21 and on the interactions that this residue forms with the environment. The importance of the protonation state of His 21 on the stability of the KH fold was addressed and validated by experimental results.

Carrier Proteins↗

PubMed2000

Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean.

Calmodulin-like domain protein kinases (CDPKs) represent a new class of calcium-dependent protein-phosphorylating enzymes that are not activated by calmodulin or phospholipid compounds. They have been found exclusively in plant and protozoal tissues. CDPKs are typified by four distinct domains: an N-terminal leader sequence, a protein kinase (PK) domain, a calmodulin-like domain (CLD), and a junction domain (JD) between the PK domain and CLD. Structural characterization of the CLD of CDPKalpha from soybean was undertaken based on the amino acid sequence homology of CLD to the structurally well-characterized calmodulin (CaM) family of structures. Tertiary models of apo-CLD, Ca(2+)-CLD complex, and intermolecularly bound Ca(2+)-CLD-JD complexes were obtained via automated and non-automated homology building methods. The resulting structures were compared and validated based on energy differences, phi-psi angle distribution, solvent accessibility, and hydrophobic potential. Circular dichroism, one-dimensional, and two-dimensional nuclear magnetic resonance spectroscopy studies of the CLD and peptides encompassing the JD provide experimental support to the models. The results suggest that there is a possible interaction between the CLD and JD domain similar to that of the CaM/calmodulin-dependent protein kinase II system. At low Ca(2+) levels, the JD may act as an autoinhibitory domain for kinase activity, and during calcium activation an intramolecular CLD-JD complex may form, relieving inhibition of the PK domain. Interactions between the JD and the C terminus of the CLD appear to be particularly important. The outcome of this study supports an intramolecular binding model for calcium activation of CDPK, although not exclusively.

Amino Acid Sequence↗

PubMed1999

Conformation in solution and dynamics of a structurally constrained linear insect kinin pentapeptide analogue.

The preferred conformations of the active diuretic insect kinin pentapeptide analogue Phe-Phe-Aib-Trp-Gly-NH2 were studied using nmr spectroscopy and molecular modeling. Structure sets consistent with rotating frame nuclear Overhauser effect spectroscopy distance constraints obtained by restrained simulated annealing in vacuo indicate a predominant population of a type II beta-turn involving the Phe1-Trp4 region. An equilibrium between this type II and a type I beta-turn formed by residues Phe2 and Gly5 was observed in a 5 ns restrained molecular dynamics simulation using the implicit generalized Born solvent accessible surface area (GB/SA) solvation model. When subjected to 500 ps dynamics with explicit water both beta-turn folds were conserved throughout the simulations. The results obtained with implicit and explicit solvation models are compared, and their consistency with the nmr observations is discussed. The behavior of the linear pentapeptide in this study is in agreement with an earlier report on the consensus conformation of the insect kinin active core derived from analysis of cyclic active analogues.

Animals↗

PubMed1999

Free energy based populations of interconverting microstates of a cyclic peptide lead to the experimental NMR data.

Analysis of nuclear Overhauser enhancement (NOE) intensities data of interconverting microstates of a peptide is a difficult problem in nmr. A new statistical mechanics methodology has been proposed recently, consisting of several steps: (1) potential energy wells on the energy surface of the molecule are identified (the corresponding regions are called wide microstates); (2) each wide microstate is then spanned by a Monte Carlo (MC) or molecular dynamics simulation starting from a representative structure, and the corresponding relative populations are obtained from the free energy calculated with the local states method; and (3) the overall NOEs and 3J coupling constants are obtained as averages over the corresponding contributions of the samples, weighted by the populations. Extending this methodology to cyclic peptides, we are treating here the hexapeptide cyclo(D-Pro1-Phe2-Ala3-Ser4-Phe5-Phe6) in DMSO, which was studied by Kessler et al. using nmr (Journal of the American Chemical Society, 1992, Vol. 114, pp. 4805-4818). They found that at least two structures are required to explain their NOE data, a conclusion also corroborated by our analysis (Journal of the American Chemical Society, 1998, Vol. 120, pp. 800-812) and led to a novel derivation of atomic solvation parameters (ASPs) for DMSO. Thus, the overall interactions within the peptide-solvent system are described approximately by Etot = EGRO + summation operator sigmaiAi, where EGRO is the energy of the GROMOS force field, Ai is the solvent-accessible surface area of atom i, and sigmai is the ASP. In the present paper the validity of these ASPs within the framework of the entire methodology is verified. This requires taking into account 23 microstates. A very good agreement is obtained between experimental and calculated NOEs and 3J coupling constants. The free energy based populations lead to the best results, which means that entropic effects should not be ignored. We have also studied the behavior of the internal angular fluctuations of the proton-proton vectors and discovered that they have a negligible effect on the calculated NOEs; this is due to the relatively concentrated wide microstates spanned by the MC simulations. The applicability of our ASPs to other cyclic peptides in DMSO is being studied in another work and preliminary results are discussed.

Algorithms↗

PubMed2002

EPR approaches to ion channel structure and function.

The fundamental processes that underlie ion channel function are permeation/ selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labelling/ electron paramagnetic resonance, EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. As an ideal test channel, we have focused our attention on the K+ channel from Streptomyces lividans, KcsA. Through site-directed spin labelling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within KcsA with high reactivity and specificity. EPR spectroscopy analysis of the spin labelled mutants yields two types of structural information: (1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and (2) distances between pairs of nitroxides through dipole-dipole interactions. Using this approach, we analysed the correlation between KcsA crystal structure and the EPR data, extend it to derive low-resolution folds of full-length KcsA and apply it in the determination of the molecular rearrangements responsible for pH-dependent gating.

Bacterial Proteins↗

PubMed2000

Discriminating between homodimeric and monomeric proteins in the crystalline state.

Scores calculated from intermolecular contacts of proteins in the crystalline state are used to differentiate monomeric and homodimeric proteins, by classification into two categories separated by a cut-off score value. The generalized classification error is estimated by using bootstrap re-sampling on a nonredundant set of 172 water-soluble proteins whose prevalent quaternary state in solution is known to be either monomeric or homodimeric. A statistical potential, based on atom-pair frequencies across interfaces observed with homodimers, is found to yield an error rate of 12.5%. This indicates a small but significant improvement over the measure of solvent accessible surface area buried in the contact interface, which achieves an error rate of 15.4%. A further modification of the latter parameter relating the two most extensive contacts of the crystal results in an even lower error rate of 11.1%.

Crystallization↗

PubMed2001

Conserved key amino acid positions (CKAAPs) derived from the analysis of common substructures in proteins.

An all-against-all protein structure comparison using the Combinatorial Extension (CE) algorithm applied to a representative set of PDB structures revealed a gallery of common substructures in proteins (http://cl.sdsc.edu/ce.html). These substructures represent commonly identified folds, domains, or components thereof. Most of the subsequences forming these similar substructures have no significant sequence similarity. We present a method to identify conserved amino acid positions and residue-dependent property clusters within these subsequences starting with structure alignments. Each of the subsequences is aligned to its homologues in SWALL, a nonredundant protein sequence database. The most similar sequences are purged into a common frequency matrix, and weighted homologues of each one of the subsequences are used in scoring for conserved key amino acid positions (CKAAPs). We have set the top 20% of the high-scoring positions in each substructure to be CKAAPs. It is hypothesized that CKAAPs may be responsible for the common folding patterns in either a local or global view of the protein-folding pathway. Where a significant number of structures exist, CKAAPs have also been identified in structure alignments of complete polypeptide chains from the same protein family or superfamily. Evidence to support the presence of CKAAPs comes from other computational approaches and experimental studies of mutation and protein-folding experiments, notably the Paracelsus challenge. Finally, the structural environment of CKAAPs versus non-CKAAPs is examined for solvent accessibility, hydrogen bonding, and secondary structure. The identification of CKAAPs has important implications for protein engineering, fold recognition, modeling, and structure prediction studies and is dependent on the availability of structures and an accurate structure alignment methodology. Proteins 2001;42:148-163.

Algorithms↗

PubMed2001

Molecular dynamics simulation reveals sequence-intrinsic and protein-induced geometrical features of the OL1 DNA operator.

We have carried out molecular dynamics simulation of the lambda OL1 DNA operator on the free and the protein-bound forms. Our results lead us to conclude that the binding of the repressor actually makes the N-7 atom of Gua8' more solvent exposed, thereby enhancing its reactivity to chemical methylation. This increase in solvent accessibility surface area occurs simultaneously with the formation of hydrogen bonds between Lys-4 of the nonconsensus flexible N-terminal arm and Gua6' of the nonconsensus half-site operator DNA. Calculations of protein--DNA interaction energies reveal that among the residues of the arm, Lys-4 contributes the most favorably to the interaction energies. This result is consistent with mutagenesis studies that established that lysine at position 4 is absolutely required for tight binding. We find that the nonconsensus arm and the nonconsensus monomer interacts less favorably with DNA than do their respective counterparts of the consensus monomer. Moreover, the six-residue flexible arm accounts for at least half the total protein--DNA interactions energy. These results are in agreement with previous experimental studies. In accord with the diffuse electron density map observed in crystallographic studies of the nonconsensus flexible arm, we find that our model built for this region is more flexible and exhibits more conformations than its consensus counterpart. The simulation also reveals that DNA bending observed near the outer edge of the operator site is an intrinsic sequence-dependent property. By contrast, the DNA-bending features observed toward the center of the operator are induced by the protein. On the whole, stepwise protein-induced bending is more pronounced in the consensus half-site operator. We also find that the unusually large helical twist (49 degrees ) observed in the protein-bound form near the center of the operator results from the binding of the protein at a base step with some propensity for high twists.

Bacteriophage lambda↗

PubMed2001

A helical arrangement of beta-substituents of dehydropeptides: synthesis and conformational study of sequential nona- and dodecapeptides possessing (Z)-beta-(1-naphthyl)dehydroalanine residues.

Sequential nona- and dodecapeptides possessing three and four (Z)-beta -(1-naphthyl)dehydroalanine (Delta(Z)Nap) residues, Boc-(L-Ala-Delta(Z)Nap-L-Leu)(n)-OCH(3) (n = 3 and 4; Boc = t-butoxycarbonyl), were synthesized to design a rigid 3(10)-helical backbone for a regular arrangement of functional groups using dehydropeptides. Their solution conformations were investigated by NMR and CD analyses, and theoretical energy calculations. Both peptides were found to adopt a 3(10)-helical conformation in CDCl(3) from their nuclear Overhauser effect spectroscopy (NOESY) spectra, which showed intense cross peaks for N(i)H-N(i+1)H proton pairs, but no cross peaks for C(alpha)(i)H-N(i+4)H pairs. The predominance of a 3(10)-helix was also supported by solvent accessibility of NH resonances. CD spectra of both peptides in tetrahydrofuran showed strong exciton couplets at around 228 nm assignable to naphthyl side chains, which are regularly arranged along a right-handed helical backbone. Chain-length effects on conformational preference in sequential peptide -(Ala-Delta(Z)Nap-Leu)(n)- were discussed based on spectroscopic analysis, energy minimization, and molecular dynamics simulations. Consequently, the repeating number n > or = 3 forms predominantly a right-handed 3(10)-helical conformation. The energy calculation also revealed that the midpoint naphthyl groups of peptide n = 4 are highly restricted to one stable orientation. In conclusion, beta-substituted alpha,beta-dehydroalanine is expected to be a unique tool for designing a rigid molecular frame of 3(10)-helix along which beta-functional groups are regularly arranged in a specific manner.

Alanine↗

PubMed2001

Solution structure of the cytoplasmic linker between domain III-S6 and domain IV-S1 (III-IV linker) of the rat brain sodium channel in SDS micelles.

The solution structure of the 36-mer peptide MP-5A in SDS micelles was investigated by CD and (1)H-NMR spectroscopies. The MP-5A was dissected from the cytoplasmic linker (K1482-A1517) connecting domain III-segment 6 (IIIS6) and domain IV-segment 1 (IVS1; III-IV linker) of the rat brain type IIA sodium channel. The molecular energy calculations including nuclear Overhauser effect and dihedral angle restraints gave a well-converged set of the structures of MP-5A for the region between I1488 and S1506. It was found that a large hydrophobic cluster is formed by I1488-F1489-M1490 (IFM motif), Y1497-Y1498, and M1501, which may be related to the fast inactivation process of the sodium channel. The solvent-accessible surface area of the IFM motif (195 A(2)), which is known to work essentially as an inactivation gate particle to occlude the ion permeation pore, gave the free energy (DeltaG) of stabilization of -3.9 kcal mol(-1) as a result of the hydrophobic interactions with its receptor. This value agreed well with the free energy of binding (inactivation) of -4.1 kcal mol(-1) calculated for the equilibrium between the open and the inactivated states of the sodium channels. It is concluded that the fast inactivation of the sodium channel is achieved by the environmental polarity-dependent conformational switching at the IFM motif, in response to the voltage-dependent activation and the movement of the S4 segments of the sodium channel.

Amino Acid Sequence↗