Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.     

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.     

The human prion protein alpha2 helix: a thermodynamic study of its conformational preferences

We have synthesized both free and terminally-blocked peptide corresponding to the second helical region of the globular domain of normal human prion protein, which has recently gained the attention of structural biologists because of a possible role in the nucleation process and fibrillization of prion protein. The profile of the circular dichroism spectrum of the free peptide was that typical of alpha-helix, but was converted to that of beta-structure in about 16 h. Instead, below 2.1 x 10(-5) M, the spectrum of the blocked peptide exhibited a single band centered at 200 nm, unequivocally associated to random conformations, which did not evolve even after 24 h. Conformational preferences of this last peptide have been investigated as a function of temperature, using trifluoroethanol or low-concentration sodium dodecyl sulfate as alpha- or beta-structure inducers, respectively. Extrapolation of free energy data to zero concentration of structuring agent highlighted that the peptide prefers alpha-helical to beta-type organization, in spite of results from prediction algorithms. However, the free energy difference between the two forms, as obtained by a thermodynamic cycle, is subtle (roughly 5-8 kJ mol(-1) at any temperature from 280 K to 350 K), suggesting conformational ambivalence. This result supports the view that, in the prion protein, the structural behavior of the peptide is governed by the cellular microenvironment.     

Stabilization of the ribonuclease S-peptide alpha-helix by trifluoroethanol

The effects of trifluoroethanol (TFE) on the stability of the alpha-helix formed by ribonuclease S-peptide, residues 1-19 of ribonuclease A, were studied by measuring circular dichroism as a function of TFE concentration, pH, and temperature. The S-peptide forms an unusually stable alpha-helix, which is known to be stabilized by TFE. The magnitude of the effect of charged groups on the peptide, manifested by the change in alpha-helix stability as a function of pH, was not altered significantly by either TFE concentration or temperature, indicating that the lower dielectric constant of TFE is not important in the stabilization of this alpha-helix. This suggests that the alpha-helix might be stabilized by many interactions in addition to the effects of charges. The titration curve of circular dichroism vs. TFE concentration appears to be cooperative at 0 degree C, but becomes progressively less cooperative at temperatures between 25 and 75 degrees C. The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

Continuum electrostatics of the C-peptide: anatomy of the problem.

A computational study of the role of all ionizable groups of the C-peptide in its helix-coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix-coil equilibrium of the C-peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix-coil transition due to the side chains of the C-peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix-coil transition of the C-peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed.     

Quantitative analysis of cyclic beta-turn models.

The beta-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the alpha-helix and beta-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of beta-turns. Recently, it has been reported (Hollósi, M., Kövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527-1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772-9784) that some pseudohexapeptides (e.g., the cyclo[(delta)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II beta-turns, although the X-ray-determined conformation was an ideal type I beta-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above beta-turn models has an influence on their conformational properties. Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II beta-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 beta-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major beta-turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.     

Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins.

Apolipophorin-III (apoLp-III) from the insect, Manduca sexta, is a 166-residue exchangeable apolipoprotein that plays a critical role in the dynamics of plasma lipoprotein interconversions. Our previous work indicated that a 36-residue C-terminal peptide fragment, generated by cyanogen bromide digestion of apoLp-III, was unable to bind to lipid surfaces (Narayanaswami V, Kay CM, Oikawa K, Ryan RO, 1994, Biochemistry 33:13312-13320), and showed no secondary structure in aqueous solution. In this paper, we have performed structural studies of this peptide (E131-Q166) complexed with SDS detergent micelles, or in the presence of the helix-inducing solvent trifluoroethanol (TFE), by two-dimensional 1H NMR spectroscopy. The peptide adopts an alpha-helical structure in the presence of both SDS and 50% TFE. The lipid-bound structure of the peptide, generated from the NMR NOE data, showed an elongated, slightly curved alpha-helix. Despite its high alpha-helix forming propensity, the peptide requires alpha helix-promoting environment to adopt an alpha-helical structure. This indicates the importance of the surrounding chemical environment and implies that, in the absence of lipid, tertiary contacts in the folded protein play a role in maintaining its structural integrity. Furthermore, the data suggest that the amphipathic helix bundle organization serves as a prerequisite structural motif for the reversible lipoprotein-binding activity of M. sexta apoLp-III.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

On the formation of the double helix between adenine single strands at acidic pH from synchrotron radiation circular dichroism experiments

Here, we present synchrotron radiation circular dichroism spectra for a series of DNA adenine strands, (dA)(n) , n = 2-10, 15, at acidic pH. Reference spectra of a protonated single strand, (dAH(+) )(n) , and a protonated double helix, (dAH(+) )(n) :(dAH(+) )(n) , are provided in the wavelength region from 175 to 330 nm. The largest spectral difference between single and double strands is in the vacuum ultraviolet, where a band changes sign. This new spectral feature that characterizes double helix formation may be useful for analytical purposes but also for shedding light on the underlying complexation mechanism. Furthermore, we find that a minimum of eight or nine bases in a strand is needed for two (dAH(+) )(n) strands to form a duplex. This is a relatively high number as compared with guanine quadruplex and cytosine i-motif formation, which is likely linked to the significant Coulomb repulsion between the all-protonated bases. Finally, spectra recorded as a function of time after sample preparation indicates that the equilibrium is slowly reached, in certain cases taking an hour or more.     

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.     

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.     

Solution structure and dynamics of bovine beta-lactoglobulin A

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine beta-lactoglobulin A at pH 2.0 and 45 degrees C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight-stranded continuous antiparallel beta-barrel and one major alpha-helix, is similar to the X-ray dimeric structure obtained at pH 6.2, including betaI-strand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. [1H]-15N NOE revealed that betaF, betaG, and betaH strands buried under the major alpha-helix are rigid on a pico- to nanosecond time scale and also emphasized rapid fluctuations of loops and the N- and C-terminal regions.     

Polypeptide models of elastin: CD and NMR studies on synthetic poly(X-Gly-Gly).

Poly(X-Gly-Gly), simple structural models for the hydrophobic, proline-devoid, regions of elastin, have been synthesized and studied by circular dichroism and NMR spectroscopies. The results gave evidence of type II beta-turns as the only ordered structure present in the polymers. The stability of the turns has been shown to decrease on hydration and to increase in the series Leu less than Ala less than Val less than Ile.     

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1.

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.     

Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.

The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds.     

The CXXC motif at the N terminus of an alpha-helical peptide

An active site containing a CXXC motif is always found in the thiol-disulphide oxidoreductase superfamily. A survey of crystal structures revealed that the CXXC motif had a very high local propensity (26.3 +/- 6.2) for the N termini of alpha-helices. A helical peptide with the sequence CAAC at the N terminus was synthesized to examine the helix-stabilizing capacity of the CXXC motif. Circular dichroism was used to confirm the helical nature of the peptide and study behavior under titration with various species. With DTT, a redox potential of E(o) = -230 mV was measured, indicating that the isolated peptide is reducing in nature and similar to native human thioredoxin. The pK(a) values of the individual Cys residues could not be separated in the titration of the reduced state, giving a single transition with an apparent pK(a) of 6.74 (+/-0.06). In the oxidized state, the N-terminal pK(a) is 5.96 (+/-0.05). Analysis of results with the modified helix-coil theory indicated that the disulfide bond stabilized the alpha-helical structure by 0.5 kcal/mol. Reducing the disulfide destabilizes the helix by 0.9 kcal/mol.     

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.     

An alternative to Urry's model for the gramicidin transmembrane channels

It is suggested that the conducting transmembrane channels formed by the linear gramicidins A, B and C may be head-to-head (formyl end-to-formyl end) dimers of double-stranded parallel β-helices. This possibility is not in conflict with any of the various experimental findings bearing on the molecular organization of the channels and represents a plausible alternative to Urry's model of a head-to-head dimer of single-stranded β^6.3-helices.     

N- and C-capping preferences for all 20 amino acids in alpha-helical peptides.

We have determined the N- and C-capping preferences of all 20 amino acids by substituting residue X in the peptides NH2-XAKAAAAKAAAAKAAGY-CONH2 and in Ac-YGAAKAAAAKAAAAKAX-CO2H. Helix contents were measured by CD spectroscopy to obtain rank orders of capping preferences. The data were further analyzed by our modified Lifson-Roig helix-coil theory, which includes capping parameters (n and c), to find free energies of capping (-RT ln n and -RT ln c), relative to Ala. Results were obtained for charged and uncharged termini and for different charged states of titratable side chains. N-cap preferences varied from Asn (best) to Gln (worst). We find, as expected, that amino acids that can accept hydrogen bonds from otherwise free backbone NH groups, such as Asn, Asp, Ser, Thr, and Cys generally have the highest N-cap preference. Gly and acetyl group are favored, as are negative charges in side chains and at the N-terminus. Our N-cap preference scale agrees well with preferences in proteins. In contrast, we find little variation when changing the identity of the C-cap residue. We find no preference for Gly at the C-cap in contrast to the situation in proteins. Both N-cap and C-cap results for Tyr and Trp are inaccurate because their aromatic groups affect the CD spectrum. The data presented here are of value in rationalizing mutations at capping sites in proteins and in predicting the helix contents of peptides.