Improved prediction for N-termini of alpha-helices using empirical information

The prediction of the secondary structure of proteins from their amino acid sequences remains a key component of many approaches to the protein folding problem. The most abundant form of regular secondary structure in proteins is the alpha-helix, in which specific residue preferences exist at the N-terminal locations. Propensities derived from these observed amino acid frequencies in the Protein Data Bank (PDB) database correlate well with experimental free energies measured for residues at different N-terminal positions in alanine-based peptides. We report a novel method to exploit this data to improve protein secondary structure prediction through identification of the correct N-terminal sequences in alpha-helices, based on existing popular methods for secondary structure prediction. With this algorithm, the number of correctly predicted alpha-helix start positions was improved from 30% to 38%, while the overall prediction accuracy (Q3) remained the same, using cross-validated testing. Although the algorithm was developed and tested on multiple sequence alignment-based secondary structure predictions, it was also able to improve the predictions of start locations by methods that use single sequences to make their predictions. Furthermore, the residue frequencies at N-terminal positions of the improved predictions better reflect those seen at the N-terminal positions of alpha-helices in proteins. This has implications for areas such as comparative modeling, where a more accurate prediction of the N-terminal regions of alpha-helices should benefit attempts to model adjacent loop regions. The algorithm is available as a Web tool, located at http://rocky.bms.umist.ac.uk/elephant.     

Flexibility of beta-sheets: principal component analysis of database protein structures

Protein folds are built primarily from the packing together of two types of structures: alpha-helices and beta-sheets. Neither structure is rigid, and the flexibility of helices and sheets is often important in determining the final fold (e.g., coiled coils and beta-barrels). Recent work has quantified the flexibility of alpha-helices using a principal component analysis (PCA) of database helical structures (J. Mol. Bio. 2003, 327, pp. 229-237). Here, we extend the analysis to beta-sheet flexibility using PCA on a database of beta-sheet structures. For sheets of varying dimension and geometry, we find two dominant modes of flexibility: twist and bend. The distributions of amplitudes for these modes are found to be Gaussian and independent, suggesting that the PCA twist and bend modes can be identified as the soft elastic normal modes of sheets. We consider the scaling of mode eigenvalues with sheet size and find that parallel beta-sheets are more rigid than antiparallel sheets over the entire range studied. Finally, we discuss the application of our PCA results to modeling and design of beta-sheet proteins.     

Influence of strand number on antiparallel beta-sheet stability in designed three- and four-stranded beta-sheets

We describe experiments that probe whether antiparallel beta-sheet secondary structure becomes more stable as the number of strands increases. Several groups, including ours, have explored this issue with peptides designed to adopt three-stranded beta-sheet conformations, but the conclusions have not been consistent. In this study, we examine the effect on conformational stability of beta-sheet lengthening perpendicular to the strand direction via analysis of designed peptides that adopt three-stranded or four-stranded antiparallel beta-sheet conformations in aqueous solution. The findings reported here, along with the context provided by earlier studies, suggest that antiparallel beta-sheet does, in general, become more stable when the number of strands is increased from two to three. We show that this conclusion is not influenced by the rigidity of the loop segment used to link adjacent beta-strands (D-Pro-Gly versus Asn-Gly). We show that further extension, from three strands to four, leads to a further increase in antiparallel beta-sheet stability.     

Secondary structure of peptides: 8. 13C n.m.r. CP/MAS investigation of solid poly(l-leucines) and poly(d-norvalines) prepared from N-carboxyanhydrides

¹³C n.m.r. CP/MAS spectra (50.3 and 75.4 MHz) of solid poly(l-lleucines) and poly(d-norvalines) measured with suitable acquisition parameters allow quantification of the composition of the secondary structure. The optimum acquisition parameters were found by systematic variation of the contact time by means of samples containing 5?0% α-helix structure. The polypeptides were prepared by primary or tertiary amine-initiated polymerizations of the corresponding amino acid NCAs and the average degrees of polymerization ($\text{DP}$) were determined by ¹H n.m.r. endgroup analysis. The mole fraction of α-helices increases with increasing $\text{DP}$; it depends on the nature of the solvent and to a lesser degree on the polymerization temperature. When prepared under identical conditions, poly(d-norvaline) samples contain more β-sheet structure than poly(l-leucine. Reprecipitation increases the α-helix content, demonstrating that a part of the original β-sheet structure is thermodynamically unstable. The presence of oligomers of $\text{DP}$ ?10 is mainly responsible for the thermodynamically stable part of the β-sheet structure. The chain growth mechanism is discussed.     

Physical reasons for secondary structure stability: alpha-helices in short peptides

It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, ^1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure^12–19 based on an extension of the conventional Zimm-Bragg model.²⁰ Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C- and S-peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;^13–19 some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α-helical “embryos” in unfolded protein chains.     

Deuterium exchange of alpha-helices and beta-sheets as monitored by electrospray ionization mass spectrometry.

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI-MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several alpha-helical peptides and beta-sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, alpha-helical, or beta-sheet structure. For 3 isomeric 31-residue alpha-helical peptides, the number of slowly exchanging hydrogens as measured by ESI-MS in 50% CF3CD2OD (pD 9.5) provided estimates of their alpha-helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of beta-sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI-MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI-MS requires much less sample (1-50 micrograms), much shorter analysis time (10-90 min), and no chemical quenching of the exchange reaction.     

Interactions between hydrophobic side chains within alpha-helices.

The thermodynamic basis of helix stability in peptides and proteins is a topic of considerable interest. Accordingly, we have computed the interactions between side chains of all hydrophobic residue pairs and selected triples in a model helix, using Boltzmann-weighted exhaustive modeling. Specifically, all possible pairs from the set Ala, Cys, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val were modeled at spacings of (i, i + 2), (i, i + 3), and (i, i + 4) in the central turn of a model poly-alanyl alpha-helix. Significant interactions--both stabilizing and destabilizing-- were found to occur at spacings of (i, i + 3) and (i, i + 4), particularly in side chains with rings (i.e., Phe, Tyr, Trp, and His). In addition, modeling of leucine triples in a helix showed that the free energy can exceed the sum of pairwise interactions in certain cases. Our calculated interaction values both rationalize recent experimental data and provide previously unavailable estimates of the constituent energies and entropies of interaction.     

Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability.

The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.     

Circular dichroism of the parallel β helical proteins pectate lyase C and E

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel β-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel β-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel β-sheets these proteins provide a unique opportunity to study the effect of parallel β-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca²⁺, derive the parallel β-helical components of the spectra, and compare these results with previous CD studies of parallel β-sheet structure. The shape and intensity of the parallel β-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel β-helical folding motif. © 1995 Wiley-Liss, Inc.     

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.     

Refolding molecular dynamics simulations of small- and middle-sized proteins in an explicit solvent

In order to elucidate the protein folding problem, we performed molecular dynamics simulations for small- and middle-sized two unfolding and six refolding proteins in an explicit solvent. Histidine-containing phosphocarrier protein and small designed protein were chosen for the simulations. We found that the protein folding process of these proteins was divided into three phases: an α -helix formation phase, a packing phase and a β -sheet formation phase. In the α -helix formation phase, an α -helix was developed from a β -turn structure through a 3₁₀-helix state. In the packing phase, proteins became compact, and tertiary structures (α / α or pre- β / β packing) were formed. Formation of a hydrophobic nucleus occurred concomitant with the α -helix formation and packing phase. Finally, in the β -sheet formation phase, a β -sheet was developed owing to the sequential formation of hydrogen bonds between two neighbouring strands, just like a "closing zipper".     

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.     

Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.     

Structural exploration of viral matrix protein 40 interaction with the transition metal ions (Ag+ and Cu2+)

Here, a theoretical and comprehensive study of the structural features and interaction properties of viral protein 40 is being briefed out to understand the mechanism of Ebola virus (EV) with structural and orbital analysis. In general, viral protein 40 is the key protein for the oligomerization, the N-terminal loop region in the viral protein 40 and it is essential for the viral replication in Ebola. The electronic structures of native N-terminal loop (His124-Asn134) and metalized (Mⁿ⁺=Ag⁺ and Cu²⁺) complexes are optimized at the M06-2X/LANL2DZ level of theory. Among Mⁿ⁺-interacted N-loop complexes, Cu²⁺-interacted N-terminal loop complex has the highest interaction energy of –973.519?kcal/mol and also it has the stabilization energy in the range of 9.92?kcal/mol. The cation-π interactions between His124, Pro131 and Arg134 residues are the important factor, which enhances the interaction energy of viral protein 40. Due to the chelation behavior of metal ions, the backbone and the side chains of N-terminal loop regions are deviated from the planarity that results in the formation of classical hydrogen bonds between N-terminal loop regions. Molecular dynamics simulation studies also revealed that the structural transformations of Nloop into a stable α-helix and β-sheet folded conformations due to the interaction of Ag⁺ and Cu²⁺ ions in the N-terminal loop region. The hydrogen bond formation and hydrophobic interactions are responsible for the stability and structural changes in N-terminal loop region. Therefore, it is clear that interaction of metal ion with viral protein-40 reduces the replication of the disease by inducing the secondary structural changes.

Communicated by Ramaswamy H. Sarma     

Analyses of circular dichroism spectra of membrane proteins

Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.     

Secondary structure of globulins from plant seeds: a re-evaluation from circular dichroism measurements

The secondary structure parameters of plant seed globulins (11S from Brassica napus L, 11S from Helianthus annuus L, IIS from Vicia faba, 7S from Phaseolus vulgaris L) have been determined from their circular dichroism spectra by the method of Provencher and Glöckner. According to this method, the proteins contain 40–50% β-sheet structure and only about 10% helical structure. We conclude, therefore, that the plant seed globulins belong to the class of β-sheet proteins. Their overall secondary structure is homologous. It is shown that the method of Provencher and Glöckner provides reasonable secondary structure parameters for proteins which are rich in β-sheet structure even if the spectral range utilized for analysis is restricted to 210–240 nm.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.