Proline in alpha-helix: stability and conformation studied by dynamics simulation

Free-energy simulations have been used to estimate the change in the conformational stability of short polyalanine alpha-helices when one of the alanines is replaced by a proline residue. For substituting proline in the middle of the helix the change in free energy of folding (delta delta G degrees) was calculated as 14 kJ/mol (3.4 kcal/mol), in excellent agreement with the one available experimental value. The helix containing proline was found to be strongly kinked; the free energy for reducing the angle of the kink from 40 degrees to 15 degrees was calculated, and found to be small. A tendency to alternate hydrogen bonding schemes was observed in the proline-containing helix. These observations for the oligopeptide agree well with the observation of a range of kink angles (18-35 degrees) and variety of hydrogen bonding schemes, in the rare instances where proline occurs in helices in globular proteins. For substituting proline at the N-terminus of the helix the change in free energy of folding (delta delta G degrees) was calculated as -4 kJ/mol in the first helical position (N1) and +6 kJ/mol in the second helical position (N2). The observed frequent occurrence of proline in position N1 in alpha-helices in proteins therefore has its origin in stability differences of secondary structure. The conclusion reached here that proline may be a better helix former in position N1 than (even) alanine, and thus be a helix initiator may be testable experimentally by measurements of fraction helical conformation of individual residues in oligopeptides of appropriate sequence. The relevance of these results in regards to the frequent occurrence of proline-containing helices in certain membrane proteins is discussed.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Conformational dynamics of helix S6 from Shaker potassium channel: simulation studies

Prolines in transmembrane (TM) alpha-helices are believed to play an important structural and/or functional role in membrane proteins. At a structural level a proline residue distorts alpha-helical structure due to the loss of at least one stabilizing backbone hydrogen bond, and introduces flexibility in the helix that may result in substantial kink and swivel motions about the effective "hinge." At a functional level, for example in Kv channels, it is believed that proline-induced molecular hinges may have a direct role in gating, i.e., the conformational change linked to opening/closing the channel to movement of ions. In this article we study the conformational dynamics of the S6 TM helix from of the Kv channel Shaker, which possesses the motif PVP--a motif that is conserved in Kv channels. We perform multiple molecular dynamics simulations of single S6 helices in a membrane-mimetic environment in order to effectively map the kink-swivel conformational space of the protein, exploiting the ability of multiple simulations to achieve greater sampling. We show that the presence of proline locally perturbs the helix, disrupting local dihedral angles and producing local twist and unwinding in the region of the hinge--an effect that is relaxed with distance from the PVP motif. We furthermore show that motions about the hinge are highly anisotropic, reflecting a preferred region of kink-swivel conformation space that may have implications for the gating process.     

Geometry of proline-containing alpha-helices in proteins.

Crystal structure analysis of proline-containing alpha-helices in proteins has been carried out. High resolution crystal structures were selected from the Protein Data Bank. Apart from the standard internal parameters, some parameters which are specifically related to the bend in the helix due to proline have been developed and analyzed. Finally the position and nature of these helices and their interactions with the rest of the protein have been analyzed.     

The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.     

Structural and functional implications of a proline residue in the antimicrobial peptide gaegurin.

Although it is commonly known as a helix breaker, proline residues have been found in the alpha-helical regions of many peptides and proteins. The antimicrobial peptide gaegurin displays alpha-helical structure and has a central proline residue (P14). The structure and activity of gaegurin and its alanine derivative (P14A) were determined by various spectroscopic methods, restrained molecular dynamics, and biological assays. Both P14 and P14A exhibited cooperative helix formation in solution, but the helical stability of P14 was reduced substantially when compared to that of P14A. Chemical-shift analysis indicated that both of the peptides formed curved helices and that P14 showed diminished stability in the region around the central proline. However, hydrogen-exchange data revealed remarkable differences in the location of stable amide protons. P14 showed a stable region in the concave side of the curved helix, while P14A exhibited a stable region in the central turn of the helix. The model structure of P14 exhibited a pronounced kink, in contrast to the uniform helix of P14A. Both peptides showed comparable binding affinities for negatively charged lipids, while P14 had a considerably reduced affinity for a neutral lipid. With its destabilized alpha-helix, P14 exhibited greater antibacterial activity than did P14A. Hence, electrostatic interaction between helical peptides and lipid membranes is believed to be the dominant factor for antibacterial activity. Moreover, helical stability can modulate peptide binding to membranes that is driven by electrostatic interactions. The observation that P14 is a more potent antibacterial agent than P14A implies that the helical kink of P14 plays an important role in the disruption of bacterial membranes.     

Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g? conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oγ atom of Ser and Thr and the backbone carbonyl oxygen at position i ? 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the β₂-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.     

TMKink: a method to predict transmembrane helix kinks

A hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from local sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.     

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.     

Differential backbone dynamics of companion helices in the extended helical coiled‐coil domain of a bacterial chemoreceptor

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four‐helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent‐exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin‐labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane‐distal tight turn (N‐helix), and the other carboxyl terminal (C‐helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide‐spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N‐ and C‐helices. For bilayer‐inserted receptors, EPR spectra from sites in the membrane‐distal protein‐interaction region and throughout the C‐helix were typical of well‐structured helices. In contrast, for approximately two‐thirds of the N‐helix, from its origin as the AS‐2 helix of the membrane‐proximal HAMP domain to the beginning of the membrane‐distal protein‐interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four‐helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.     

3(10)-Helix adjoining alpha-helix and beta-strand: sequence and structural features and their conservation

Does the amino acid use at the terminal positions of an α‐helix become altered depending on the context—more specifically, when there is an adjoining 3₁₀‐helix, and can a single helical cylinder encompass the resultant composite helix? An analysis of 138 and 107 cases of 3₁₀–α and α–3₁₀ composite helices, respectively, found in known protein structures indicate that the secondary structural element occurring first imposes its characteristics on the sequence of the structural element coming next. Thus, when preceded by a 3₁₀‐helix, the preference of proline to occur at the N1 position of an α‐helix is shifted to the N2 position, a typical characteristic of the C‐terminal capping of the 3₁₀‐helix. When an α‐ or a 3₁₀‐helix leads into a helix of the other type, there is a bend at the junction, especially for the 3₁₀–α composite, with the two junction residues facing inward and buried within the structure. Thus a single helical cylinder may not properly represent a composite helix, the bend providing a means for the tertiary structure to assume a globular shape, very much akin to what a proline‐induced kink does to an α‐helix. The tertiary structural context in which β–3₁₀ and 3₁₀–β composites occurs can be different, causing the angle between the secondary structural elements in the two cases to be different. Composites of 3₁₀‐helices and β‐strands are much more conserved among members in families of homologous structures than those between two types of helices; in many of the former instances, the 3₁₀‐helix constitutes the loops in β‐hairpin or β–β‐corner motifs. The overall fold of the chain may be more conserved than the actual identify of the secondary structure elements in a composite. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 147–162, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.     

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Helix geometry in proteins

In this report we describe a general survey of all helices found in 57 of the known protein crystal structures, together with a detailed analysis of 48 alpha-helices found in 16 of the structures that are determined to high resolution. The survey of all helices reveals a total of 291 alpha-helices, 71 3(10)-helices and no examples of pi-helices. The conformations of the observed helices are significantly different from the "ideal" linear structures. The mean phi, psi angles for the alpha- and 3(10)-helices found in proteins are, respectively, (-62 degrees, -41 degrees) and (-71 degrees, -18 degrees). A computer program, HBEND, is used to characterize and to quantify the different types of helix distortion. alpha-Helices are classified as regular or irregular, linear, curved or kinked. Of the 48 alpha-helices analysed, only 15% are considered to be linear; 17% are kinked, and 58% are curved. The curvature of helices is caused by differences in the peptide hydrogen bonding on opposite faces of the helix, reflecting carbonyl-solvent/side-chain interactions for the exposed residues, and packing constraints for residues involved in the hydrophobic core. Kinked helices arise either as a result of included proline residues, or because of conflicting requirements for the optimal packing of the helix side-chains. In alpha-helices where there are kinks caused by proline residues, we show that the angle of kink is relatively constant (approximately 26 degrees), and that there is minimal disruption of the helix hydrogen bonding. The proline residues responsible for the kinks are highly conserved, suggesting that these distortions may be structurally/functionally important.     

Crystal structure of human heme oxygenase-1.

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.     

1H NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A

Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata solution structure and biological activity.

The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.