Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the G- and H-helices of myoglobin

Myoglobin has been extensively studied as a model system for protein folding in vitro. As part of an ongoing study of myoglobin folding, we have synthesized a series of peptide fragments corresponding to portions of the sequence of the sperm whale protein. The conformational preferences of these peptides have been investigated by circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution. In this paper we describe the folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence. The Mb-G peptide shows evidence of a very small population of helical conformations in aqueous solution, both by CD and NMR. By contrast, the monomeric Mb-H peptide is found by CD to adopt a significant population (ca. 30%) of ordered helix and by NMR to populate helical conformations in rapid dynamic equilibrium with unfolded states. The Mb-H peptide undergoes a well-characterized, concentration-dependent monomer-tetramer equilibrium. At peptide concentrations greater than 1 mM there is an increase in the population of helix, to approximately 85% according to the CD spectrum, through self-association to form a tetramer. Both medium-range NOE connectivities and a CD spectrum characteristic of ordered helix are observed at low peptide concentrations, establishing that helical conformations are present in the monomeric state of Mb-H. The relative helicity at various sites throughout the Mb-H peptide has been estimated using a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities. The population of ordered helix is seen to be highest in the center of the peptide sequence; the ends of the peptide show evidence of pronounced fraying.     

Conformations of peptide fragments from the FK506 binding protein: comparison with the native and urea-unfolded states

The helix-forming tendency of seven peptide fragments corresponding with the entire sequence of the FK506 binding protein (FKBP) has been investigated in aqueous buffer and in 2,2,2-trifluoroethanol (TFE) using CD and NMR spectroscopy. All fragments exhibited random coil conformations in aqueous buffer, whereas the amount of helix induced in the peptide fragments by TFE varied. The fragment with the highest degree of helicity in TFE corresponded with the single (alpha-helix in native FKBP. Fragments corresponding with beta-strands 2 and 3 also exhibited strong propensity towards helix formation. In contrast, the fragment corresponding with beta-strand 1 did not form helix in TFE. The inherent helix-forming tendencies are interpreted in light of the native structure to suggest possible folding nucleation sites. Conformational sampling in each peptide fragment was also compared with that observed in urea-denatured FKBP. With the exception of the fragment corresponding with beta-strand 2, the formation of helical structures in the peptide fragments in TFE was correlated with the observation of turn and/or helix conformers in urea-unfolded FKBP. Surprisingly, peptide fragments in aqueous solution were less structured than the corresponding regions in urea-denatured FKBP. The conformational differences between the peptide fragments and unfolded FKBP were not due to the urea buffer or to differences in their rotational correlation times. We conclude that local amino acid interactions are not generally sufficient to account for the formation of non-random conformations in unfolded FKBP. Formation of non-random structures in unfolded FKBP may require stabilization of incipient turn or helical conformations through transient contact with non-local non-polar residues.     

Kinetics of peptide folding: computer simulations of SYPFDV and peptide variants in water

The folding of Ser-Tyr-Pro-Phe-Asp-Val (SYPFDV), and sequence variants of this peptide (SYPYD and SYPFD) are studied computationally in an explicit water environment. An atomically detailed model of the peptide is embedded in a sphere of TIP3P water molecules and its optimal structure is computed by simulated annealing. At distances from the peptide that are beyond a few solvation shells, a continuum solvent model is employed. The simulations are performed using a mean field approach that enhances the efficiency of sampling peptide conformations. The computations predict a small number of conformations as plausible folded structures. All have a type VI turn conformation for the peptide backbone, similar to that found using NMR. However, some of the structures differ from the experimentally proposed ones in the packing of the proline ring with the aromatic residues. The second most populated structure has, in addition to a correctly folded backbone, the same hydrophobic packing as the conformation measured by NMR. Our simulations suggest a kinetic mechanism that consists of three separate stages. The time-scales associated with these stages are distinct and depend differently on temperature. Electrostatic interactions play an initial role in guiding the peptide chain to a roughly correct structure as measured by the end-to-end distance. At the same time or later the backbone torsions rearrange due to local tendency of the proline ring to form a turn: this step depends on solvation forces and is helped by loose hydrophobic interactions. In the final step, hydrophobic residues pack against each other. We also show the existence of an off the pathway intermediate, suggesting that even in the folding of a small peptide "misfolded" structures can form. The simulations clearly show that parallel folding paths are involved. Our findings suggest that the process of peptide folding shares many of the features expected for the significantly larger protein molecules.     

Long-term results of total penile reconstruction with a prefabricated lateral arm free flap

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.     

Variability in function measurements of three sensory foot nerves in neuropathic diabetic patients

Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.     

Managed mental health care: problems and possibilities

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of synthetic peptide analogs of the sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. As previously reported (Young et al., Virology, 238, 291), a peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) inhibited syncytia formation after transfection of Cos cells with the hemagglutinin-neuraminidase and F protein cDNAs. A peptide analog which had an alanine residue in place of the first leucine residue in the zipper motif (ALDKAEESNSKLDKVNVKLT) retained inhibitory activity but less than the original peptide. Further loss in activity was observed in a peptide in which two of the leucine residues were replaced with alanine (ALDKAEESNSKADKVNVKLT), and a peptide which had all leucine residues in the zipper motif replaced with alanine (ALDKAEESNSKADKVNVKLT) had no inhibitory activity. The three-dimensional conformations of these peptides in aqueous solution were determined through the use of nuclear magnetic spectroscopy and molecular modeling. Results showed that while the wild-type peptide formed a helix with properties between an alpha-helix and a 3(10) helix with leucine residues aligned along one face of the helix, progressive substitution of leucine residues with alanine resulted in the progressive loss of helical structure. The results suggest that alterations of leucine residues in the zipper motif disrupt secondary structure of the peptide and that this structure is critical to the inhibitory activity of the peptide.     

Conformational studies by NMR of the antimicrobial peptide, drosocin, and its non-glycosylated derivative: effects of glycosylation on solution conformation

Drosocin is a cationic 19 amino acid peptide secreted by Drosophila in response to septic injury. The sequence (GKPRPYSPRPTSHPRPIRV) contains six Pro and four Arg residues which are incorporated into three repeated triplet sequences Pro-Arg-Pro. The peptide is glycosylated at Thr11 and has potent antimicrobial activity. This activity is markedly reduced on deglycosylation, but a structural basis for this has not been previously established. In the current study, the solution conformations of drosocin and its non-glycosylated derivative were determined by NMR spectroscopy and structure calculations. The NMR and structure studies showed that the peptides have significant populations of essentially random coil conformations in aqueous solution. Addition of 50% trifluoroethanol causes the development of small populations of folded conformations, mainly in the form of turns. In particular, turn elements occur near residues 4-7, 10-13, 17, and 18. No substantial difference was detected in the predominantly random coil conformation of the glycosylated and non-glycosylated forms, but there are subtle differences in the small populations of folded conformers. In particular, the turn at residues 10-13 tends toward a more extended structure on glycosylation, while there is some tightening of the downstream turn at residues 17 and 18. There are a significant number of nuclear Overhauser enhancement contacts between the sugar moiety and the peptide near the glycosylation site, consistent with a close association between them. Despite this close association, the pKa of H13, which is proximate to the glycosylation site, was found to be unaffected by glycosylation.     

A role of myofilament Ca2+ sensitivity in enhanced vascular reactivity in cardiomyopathic hamsters

Conformational studies of the salivary peptides histatin 3 (H3) and histatin 5 (H5) were performed by NMR and circular dichroism (CD) in aqueous and nonaqueous solutions. Histatin 5 has no defined structure in H2O but adopts a more helical conformation in dimethyl sulfoxide and aqueous trifluoroethanol. This is in agreement with the CD analysis, which shows no secondary structure in H2O but increasing helical content in the presence of trifluoroethanol. CD analysis shows that H3 has less propensity to form a helical structure than H5 in similar conditions. The NMR analysis of H3 in H2O at pH 7.4 reveals that its conformational mobility is less than that of H5 as indicated by the observation of backbone cross peaks alphaN (i, i + 1) and NN (i, i + 1) and the slow exchanging amide protons in the C-terminus. However, H3 remains essentially unordered as suggested by the lack of longer range nuclear Overhauser effects (NOEs) in the NOESY spectrum. H3 becomes much more ordered in a mixture of 50:50 H2O-dimethyl sulfoxide as indicated by the numerous NOEs, including several side chain to side chain and side chain to backbone connectivities. Our data suggest that in these conditions H3 contains a turn in the region of K13 to K17 and possibly a 3(10) helix at the C-terminus. This study demonstrates that H3 and H5 are both conformationally mobile and that each adopt different types of conformations in aqueous and nonaqueous solutions.     

Solution structure of a retro-inverso peptide analogue mimicking the foot-and-mouth disease virus major antigenic site. Structural basis for its antigenic cross-reactivity with the parent peptide

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is alpha-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical beta-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.     

A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H-, 13C-NMR, and mutational analysis

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.     

Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule.     

Characterization of the conformations of antigenic peptides of protein lactate dehydrogenase (LDH-C4) by electrospray ionization mass spectrometry

The conformations of several rationally designed antigenic peptides that mimic, to varying degrees, an antibody-binding region of protein lactate dehydrogenase isozyme (LDH-C4) are investigated by deuterium/hydrogen exchange and electrospray ionization mass spectrometry (ESI-MS). The approach involves monitoring the reverse-exchange of deuterium, incorporated at the labile sites in the peptides, with hydrogen as a function of time by ESI-MS. Idealized forms of a segment of the native antigen are shown to be more conformationally restricted than the native peptide based on level of deuterium that remains incorporated at the labile sites over time. From the number of amide groups of the peptide backbone that retain deuterium, estimates of the helical content of each peptide have been measured that are in close agreement with those determined by Fourier transform infrared (FTIR) spectroscopy in separate experiments. A single amino acid substitution in the idealized helical construct results in a conformational change easily detected by the deuterium exchange ESI-MS method. The approach is shown to be a viable method for characterizing the conformations of protein antigens at the local level and for screening the conformations of antigenic peptides designed to elicit optimal immune responses.     

Nursing home residents at risk of hospitalization and the characteristics of their hospital stays

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu41-58, Vpu52-74 and Vpu63-81, by a combination of 2D 1H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu41-58 has an alpha-helix from residues 42 to 50 while a second helix is found for Vpu52-74 from residues 57 to 69. Vpu63-81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two alpha-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus.     

Solution conformations and thermodynamics of structured peptides: molecular dynamics simulation with an implicit solvation model

Calculations of the ensemble of solution conformations and thermodynamics of an analogue of the C-terminal helix of ribonuclease A (RN24) and of a synthetic, beta-hairpin forming peptide (BH8) are presented. For efficient sampling of conformation space, molecular dynamics simulations with an implicit solvent potential and umbrella sampling of the potential energy are performed. Starting from the fully extended chains, the simulations yield several folding and unfolding transitions between disordered (coil) conformations of the peptides and the "native" state (RN24, helix; BH8, hairpin); the simulations also lead to the occurrence of "misfolded" conformations (RN24, hairpin; BH8, helix). In agreement with experiment, the calculations predict 58% helix for RN24 at 275 K and an antiparallel-beta content of 38% at 275 K for BH8; the calculated probabilities for the misfolded species are 2% or smaller at all temperatures considered (250-1100 K). Good agreement is also shown between the calculated 3JHNalpha spin-spin coupling constants of RN24 and BH8 at 275 K, and those obtained from NMR experiments at the same temperature. From the calculated probabilities of helix (h), beta-hairpin (b), and coil (c), the free energy differences between the structured substates are DeltaGch=Gc-Gh approximately 1 kcal/mol and DeltaGbh>/=1.8 kcal/mol for RN24, and DeltaGcb approximately 0.7 kcal/mol and DeltaGhb>/=2.7 kcal/mol for BH8. The free energy difference between "correctly" folded and misfolded secondary structures are of interest for understanding the alpha to beta transition that is thought to play a role in amyloid fibril formation.     

High populations of non-native structures in the denatured state are compatible with the formation of the native folded state

The structures of the denatured states of the spectrin SH3 domain and a mutant designed to have a non-native helical tendency at the N terminus have been analyzed under mild acidic denaturing conditions by nuclear magnetic resonance methods with improved resolution. The wild-type denatured state has little residual structure. However, the denatured state of the mutant has an approximately 50% populated helical structure from residues 2 to 14, a region that forms part of the beta-sheet structure in the folded state. Comparison with a peptide corresponding to the same sequence shows that the helix is stabilized in the whole domain, likely by non-local interactions with other parts of the protein as suggested by changes in a region far from the mutated sequence. These results demonstrate that high populations of non-native secondary structure elements in the denatured state are compatible with the formation of the native folded structure.     

The structure of Fis mutant Pro61Ala illustrates that the kink within the long alpha-helix is not due to the presence of the proline residue

The influence of proline on bending of the alpha-helix was investigated by replacement of the proline residue located in the middle of the long alpha-helix of the Fis protein with alanine, serine, or leucine. Each of the three substitutions folded into a stable protein with the same or higher melting points than the wild-type, but only Pro61Ala was functionally active in stimulating Hin-mediated DNA inversion. Pro61Ala formed crystals that were isomorphous with the wild-type protein allowing the structure to be determined at 1.9-A resolution by x-ray diffraction methods. The structure of the Pro61Ala mutant is almost identical to the wild-type protein, consistent with its near wild-type activity. One of the alpha-helices, the B-helix, is kinked in the wild-type Fis protein by 20 degrees which was previously assumed to be caused solely by the presence of proline 61 in the center of the helix. However, the B-helix is still kinked by 16 degrees when proline 61 is replaced by alanine. Local peptide backbone movement around residue 57 adjusts the geometry of the helix to accommodate the new main chain hydrogen bond between the -CO group in Glu57 and the -NH group in Ala61. Thus, the kink of the alpha-helix in Pro61Ala does not require the presence of proline.     

Direct NMR measurement of folding kinetics of a trimeric peptide

Direct NMR measurements of the folding kinetics are performed on a collagen-like triple helical peptide. The triple helical peptide was designed to model a biologically important region of collagen and has the sequence (POG)3ITGARGLAG(POG)4. Triple helical peptides were synthesized with specifically labeled 15N amino acid residues in key positions, and the kinetics of folding of the individual residues were monitored directly by measuring the loss of monomer intensity and the increase in trimer intensity. The residues at the terminal ends and central region could be followed independently and quantitated directly. Residues located at the terminal ends have rates and kinetics of folding that are distinct from residues in the central region of the peptide. This allows the monitoring of different steps in the folding mechanism and the postulation of the existence of a kinetic intermediate. The NMR data are consistent with a mechanism of association/nucleation and propagation. Hereditary connective tissue diseases are associated with mutations that result in abnormal folding of collagen, and the NMR folding experiments on a collagen-like peptide provide a basis for characterizing the molecular defect in folding mutations.     

The single helix in protein L is largely disrupted at the rate-limiting step in folding

To investigate the role of helix formation in the folding of protein L, a 62 residue alpha/beta protein, we studied the consequences of both single and multiple mutations in the helix on the kinetics of folding. A triple mutant with 11 additional carbon atoms in core residues in the amino-terminal portion of the helix folded substantially faster than wild type, suggesting that hydrophobic association with residues elsewhere in the protein occurs at the rate-limiting step in folding. However, helix-destabilizing mutations had little effect on the rate of folding; in particular, a triple glycine substitution on the solvent-exposed side of the helix increased the unfolding rate 56-fold while reducing the folding rate less than threefold. Thus, in contrast to the predictions of models of folding involving the coalescence of well-formed secondary structure elements, the single helix in protein L appears to be largely disrupted at the rate-limiting step in folding and unfolding.