Exploring the sequence patterns in the alpha-helices of proteins

This paper reports an extensive sequence analysis of the alpha-helices of proteins. alpha-Helices were extracted from the Protein Data Bank (PDB) and were divided into groups according to their sizes. It was found that some amino acids had differential propensity values for adopting helical conformation in short, medium and long alpha-helices. Pro and Trp had a significantly higher propensity for helical conformation in short helices than in medium and long helices. Trp was the strongest helix conformer in short helices. Sequence patterns favoring helical conformation were derived from a neighbor-dependent sequence analysis of proteins, which calculated the effect of neighboring amino acid type on the propensity of residues for adopting a particular secondary structure in proteins. This method produced an enhanced statistical significance scale that allowed us to explore the positional preference of amino acids for alpha-helical conformations. It was shown that the amino acid pair preference for alpha-helix had a unique pattern and this pattern was not always predictable by assuming proportional contributions from the individual propensity values of the amino acids. Our analysis also yielded a series of amino acid dyads that showed preference for alpha-helix conformation. The data presented in this study, along with our previous study on loop sequences of proteins, should prove useful for developing potential 'codes' for recognizing sequence patterns that are favorable for specific secondary structural elements in proteins.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

Intrinsic helical propensities and stable secondary structure in a membrane-bound fragment (S4) of the shaker potassium channel.

The location and stability of helical secondary structure in a fragment comprising an extended sequence of the S4 transmembrane segment of the Shaker potassium channel was determined in methanol, and when bound to vesicles composed of egg phosphatidylcholine: egg phosphatidylglycerol (4:1; mol:mol) in water. The N-acetylated, C-amidated peptide corresponds to the sequence comprising residues A355-I384 in the Shaker potassium channel. Although NOEs characteristic of helical structure encompass essentially the full peptide sequence in methanol, analysis of amide and CH(alpha) chemical shifts, and amide exchange protection factors establish that stable helical structure comprises only around the first 22 amino acids of the 30 residue peptide. This sequence corresponds to that predicted to have the highest helical stability in water, indicating that while helical structure is considerably stabilised in methanol, the relative helical propensities of amino acids in methanol may be similar to those in water.In the presence of vesicles containing negatively charged lipids, helical structure corresponding to a maximum of around 40 % of the extended S4 peptide is induced; no helical structure is induced in the presence of vesicles composed only of neutral lipids. The location of stable helical structure in the membrane-bound peptide was determined by amide hydrogen-deuterium exchange trapping, and was shown to encompass the sequence between residues near M2 and I18. This sequence is similar to that having high helix propensity in water and methanol, supporting the idea that intrinsic helical propensities are important in defining the location of stable helical structure in polypeptides bound in the interfacial region of lipid bilayers. The study defines an approach to determining the location of, and contributions to, the stability of helical secondary structure in membrane-reconstituted polypeptides.     

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.     

A CD study of the α-helix nucleation hypothesis

One of the dilemmas in predicting the secondary structure of proteins from their amino acid propensity for a given conformation is the presence of all amino acids in all types of secondary structure, regardless of their propensity for that specific structure. One explanation is the nucleation hypothesis that only a few residues with a strong propensity for the secondary structure, such as the α-helix structure, initiates its formation and propagates the structure through indifferent sequences until strong breakers terminate the growth on both ends. Eight 15-mer peptides were studied to examine the α-helix nucleation hypothesis. The nucleation sequence of VAEAK, with high helix propensity, was mixed with an indifferent sequence of TSDSR in all possible permutations. From the percent α-helix structure derived from the CD at 222 nm, it appears that helicity does not propagate through the indifferent sequence. © 1994 John Wiley & Sons, Inc.     

Molecular dynamics simulations of helix folding: The effects of amino acid substitution

Molecular dynamics simulations were applied to helix folding of alanine-based synthetic peptides. A single alanine residue in the middle of the peptide was substituted with various nonpolar amino acids (leucine, isoleucine, valine, glycine or proline) to study the effect of the substitution. Unlike many other molecular dynamics simulations, nonhelical initial conformations were used in our simulations to study the folding process. An average solvent effect was included in the energy function to simplify the solvent calculation and to overcome the multiple minima problem. During the simulations, the peptides folded into helices in nanoseconds. Compact structures containing two helical segments were also observed. The calculated helical ratios of the peptides showed the same rank order as observed experimentally for the alanine-based peptides. Within a peptide, the helical ratio of each residue was calculated and a minimum was found near the center of the sequence for all peptides. The substitutions had different asymmetric effects on the helical ratios of the residues preceding and following the substitution site, indicating different helix capping preferences of the substituting amino acids. © 1997 John Wiley & Sons, Inc. Biopoly 42: 633–644, 1997     

Investigation of penetratin peptides. Part 1. The environment dependent conformational properties of penetratin and two of its derivatives.

The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three alpha-helices and one beta-turn between helices II and III. Its third helix from the N-terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments: 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9: 1), on the basis of 553 distance restraints, the 4-12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both alpha-helical and 310-helical features, the structure of peptide 3 is predominantly a 310-helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment.     

Secondary structure of substance P bound to liposomes in organic solvents and in solution from Raman and CD spectroscopy.

The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns.     

Non‐native α‐helix formation is not necessary for folding of lipocalin: Comparison of burst‐phase folding between tear lipocalin and β‐lactoglobulin

Tear lipocalin and β‐lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non‐native helical structures are formed during the early stage of β‐lactoglobulin folding. To address whether the non‐native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped‐flow methods measuring the time‐dependent changes in circular dichroism (CD) spectrum and small‐angle X‐ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst‐phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst‐phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non‐native helix formation is not general for folding of all lipocalin family members. The non‐native helix content in the burst‐phase folding appears to depend on helical propensities of the amino acid sequence. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.     

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.     

Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an alpha-helical conformation

Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.     

Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity

It was established previously that helical propensities of different amino acid residues in the middle of α‐helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three‐dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine‐based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the α‐helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent‐exposed positions in the middle and at the C terminus of the α‐helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.     

Solution conformation of human apolipoprotein C-1 inferred from proline mutagenesis: far- and near-UV CD study

Solution structure of lipid-free apolipoprotein C-1 (apoC-1, 6.6 kD) was analyzed by circular dichroism (CD) of 15 mutants containing single Pro or Ala substitutions in predicted alpha-helical regions. While the majority of Pro substitutions induce complete (L11P, L18P, R23P, I29P, M38P, W41P, T45P) or partial (G15P, L34P) helical unfolding, similar substitutions at other sites (A7P, Q31P, V49P, L53P) do not cause large changes in the secondary structure or stability. The results suggest that lipid-free apoC-1 is comprised of two dynamic helices that are stabilized by interhelical interactions and are connected by a short linker containing residues 30-33. We propose that the minimal folding unit in the lipid-free state of this and other exchangeable apolipoproteins comprises the helix-turn-helix motif formed of four 11-mer sequence repeats. Comparison of the helical content in lipid-free and lipid-bound apoC-1 suggests that lipid binding shifts the conformational equilibrium toward preexisting highly helical conformation. Remarkably, near-UV CD spectra of wild type and mutant apoC-1 are not significantly altered upon thermal or chemical unfolding and thus result from residual aromatic clustering that is retained in the unfolded state. Correlation of far- and near-UV CD of the mutant peptides suggests that the hydrophobic cluster containing W41 is essential for the helical stability and may form a helix nucleation site in apoC-1.     

Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin

The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.     

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes

The secondary structure of alamethicin in lipid membranes below and above the lipid phase transition temperature Tt is determined by Raman spectroscopy and circular dichroism (CD) measurements. In both cases structural data are obtained by fitting the experimental spectra by a superposition of the spectra of 15 reference proteins of known three-dimensional structure. According to the Raman experiments, in a lipid bilayer above Tt alamethicin is helical from residue 1 to 12, whereas below Tt the helix extends from residue 1 to 16. The remaining C-terminal part is nonhelical up to the end residue 20 both above and below Tt. A considerable lower helix content is derived from CD, namely, 38% and 46% above and below Tt, respectively, in agreement with several reported values for CD in the literature. It is shown that the commonly used set of CD spectra of water-soluble reference proteins is unsuitable to describe the CD spectra of alamethicin correctly. Therefore the secondary structure of alamethicin as derived from CD measurements is at the present state of analysis unreliable. In contrast to the case of alamethicin, the CD spectra of melittin in lipid membranes are correctly described by the reference protein spectra. The helix content of melittin is determined thereby to be 72% in lipid membranes above Tt and 75% below Tt. The data are in accord with a structure where the hydrophobic part of melittin adopts a bent helix as determined recently by Raman spectroscopy [Vogel, H., & J?hnig, F. (1986) Biophys. J. 50, 573]. The orientational order parameters of the helical parts of alamethicin and of melittin in a lipid membrane are deduced from the difference between a corresponding CD spectrum of a polypeptide in planar multibilayers and that in lipid vesicles. The presented method for determining helix order parameters is new and may be generally applicable to other membrane proteins. The orientation of the helical part of both polypeptides depends on the physical state of the lipid bilayer at maximal membrane hydration and in the ordered lipid state furthermore on the degree of membrane hydration. Under conditions where alamethicin and melittin are incorporated in an aggregated form in a fluid lipid membrane at maximal water content the helical segments are oriented preferentially parallel to the membrane normal. Cooling such lipid membranes to a temperature below Tt changes the orientation of the helical part of alamethicin as well as melittin toward the membrane plane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational preference functions for predicting helices in membrane proteins

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the α-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in α-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 α-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known α-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements. © 1993 John Wiley & Sons, Inc.