M. Angeles Jiménez

Conformational investigation of designed short linear peptides able to fold into β-hairpin structures in aqueous solution

BACKGROUND Formation of secondary structure plays an important role in the early stages of protein folding. The conformational analysis of designed peptides has proved to be very useful for identifying the interactions responsible for the formation and stability of alpha-helices. However, very little is known about the factors leading to the formation of beta-hairpins. In order to get a good beta-hairpin-forming model peptide, two peptides were designed on the basis of beta-sheet propensities and individual statistical probabilities in the turn sites, together with solubility criteria. The conformational properties of the two peptides were analyzed by two-dimensional NMR methods. RESULTS Long-range cross-correlations observed in NOE and ROE spectra, together with other NMR evidence, show that peptide IYSNPDGTWT forms a highly populated beta-hairpin in aqueous solution with a type I beta-turn plus a G1 beta-bulge conformation in the chain-bend region. The analogous peptide with a Pro5 substituted by Ser forms, in addition to the previous conformation, a second beta-hairpin with a standard type I beta-turn conformation, and the two forms are in fast dynamic equilibrium with one another. The effect of pH demonstrates the existence of a stabilizing interaction between the Asn and Asp sidechains. The populations of beta-hairpin conformations increase in the presence of trifluoroethanol (a structure-enhancing solvent). On the other hand, some residual structure persists at a high denaturant concentration (8 M urea). CONCLUSIONS This work highlights the importance of the beta-turn residue composition in determining the particular type of beta-hairpin adopted by a peptide, though a role of interstrand sidechain interactions in the stabilization of the formed beta-hairpin is not discarded. The fact that trifluoroethanol can stabilize alpha-helices or beta-hairpins depending on the intrinsic properties of the peptide sequence is again shown. An additional example of the presence of residual structure under denaturing conditions is also presented.

Factors involved in the stability of isolated β-sheets: Turn sequence, β-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20‐residue peptide able to form a significant population of a three‐stranded up‐and‐down antiparallel β‐sheet in aqueous solution. To improve our β‐sheet model in terms of the folded population, we have modified the sequences of the two 2‐residue turns by introducing the segment DPro‐Gly, a sequence shown to lead to more rigid type II′ β‐turns. The analysis of several NMR parameters, NOE data, as well as ΔδCαH, ΔδCβ, and ΔδCβ values, demonstrates that the new peptide forms a β‐sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on β‐hairpin‐forming peptides showing the essential role of the turn sequence for β‐hairpin folding. The well‐defined β‐sheet motif calculated for the new designed peptide (pair‐wise RMSD for backbone atoms is 0.5 ± 0.1 Å) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted β‐sheet than in a flatter one. The twist observed in the up‐and‐down antiparallel β‐sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by β‐sheet twisting relative to a “flat” β‐sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.

Structural analysis of peptides encompassing all α-helices of three α/β parallel proteins: Che-Y, flavodoxin and P21-Ras: Implications for α-Helix stability and the folding of α/β parallel proteins

In an attempt to delineate the early folding events of structurally related proteins with no sequence homology, peptides including all five α-helices of three α/β parallel open-sheet proteins, Che-Y, flavodoxin and P21-ras, have been analyzed by circular dichroism (far-UV CD) and nuclear magnetic resonance (NMR) in water and 30% (v/v) trifluoroethanol (TFE). Comparison between the helical content estimations from far-UV CD and the results from the NMR analysis renders a reasonably good qualitative correlation, indicating that the same phenomenon is underlined by both methods. Helix limits, as indicated by the existence of ( i,i + 3) nuclear Overhauser effect (NOE) cross-correlations and significant up-field conformational shifts of the C α H protons, are practically coincident with those in the folded protein. On the other hand, the conformation of the side-chains differs markedly from those in the folded protein. Observation of NOE cross-correlations between pairs of residues at positions i,i + 3 has been used to statistically quantify free energies of i,i + 3 side-chain-side-chain interactions between the different pairs of residues in an α-helix. This analysis indicates that interactions between hydrophobic side-chains seem to be quite favorable for helix formation. The behaviour in aqueous solution of the structural equivalent peptides for the three proteins is quite unrelated except for the peptides corresponding to helices two and five. We postulate that, in the α/β parallel proteins, those helices that join two β-strands flanking another non-consecutive β-strand should not be stable for folding reasons.

Tryptophan residues: Scarce in proteins but strong stabilizers of β‐hairpin peptides

Tryptophan plays important roles in protein stability and recognition despite its scarcity in proteins. Except as fluorescent groups, they have been used rarely in peptide design. Nevertheless, Trp residues were crucial for the stability of some designed minimal proteins. In 2000, Trp–Trp pairs were shown to contribute more than any other hydrophobic interaction to the stability of β‐hairpin peptides. Since then, Trp–Trp pairs have emerged as a paradigm for the design of stable β‐hairpins, such as the Trpzip peptides. Here, we analyze the nature of the stabilizing capacity of Trp–Trp pairs by reviewing the β‐hairpin peptides containing Trp–Trp pairs described up to now, the spectroscopic features and geometry of the Trp–Trp pairs, and their use as binding sites in β‐hairpin peptides. To complete the overview, we briefly go through the other relevant β‐hairpin stabilizing Trp–non‐Trp interactions and illustrate the use of Trp in the design of short peptides adopting α‐helical and mixed α/β motifs. This review is of interest in the field of rational design of proteins, peptides, peptidomimetics, and biomaterials.

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of β-sheet structure in peptides is a subject of great current interest. A β-hairpin, the smallest β-sheet motif, consists of two antiparallel hydrogen-bonded β-strands linked by a loop region. We have performed a statistical analysis on protein β-hairpins showing that the most abundant types of β-hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of 13Cα and 13Cβ conformational shifts, as expected on the basis of their φ and ψ angles. This fact strongly supports the potential value of 13Cα and 13Cβ conformational shifts as a means to identify β-hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of 13Cα and 13Cβ conformational shifts in 13 short peptides, 10–15 residues long, that adopt β-hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify β-hairpin populations in peptides.

Therapeutic Index of Gramicidin S is Strongly Modulated by D-Phenylalanine Analogues at the β-Turn

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)2, with d-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several pathogenic bacteria. While all D-Phe substitutions are shown by NMR to preserve the overall beta-sheet conformation, they entail subtle structural alterations that lead to significant modifications in biological activity. In particular, the analogue incorporating D-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the D-Tic aromatic ring and the Orn delta-amino group may help explain the improved antibiotic profile of this analogue.

Sequence inversion and phenylalanine surrogates at the β-turn enhance the antibiotic activity of gramicidin S

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

Disulfide bonds versus Trp···Trp pairs in irregular β-hairpins: NMR structure of vammin loop 3-derived peptides as a case study

Where a noncovalent interaction is better than a covalent bond: The most stabilising cross‐strand pairs were incorporated into an irregular β‐hairpin, loop 3 of vammin. 1H and 13C NMR conformational analyses of these designed peptides indicated that an edge‐to‐face Trp⋅⋅⋅Trp interaction leads to a β‐hairpin that is more stable than a disulfide bond.

De novo Design of Monomeric β-Hairpin and β-Sheet Peptides

: Since the first report in 1993 (JACS 115, 5887-5888) of a peptide able to form a monomeric beta-hairpin structure in aqueous solution, the design of peptides forming either beta-hairpins (two-stranded antiparallel beta-sheets) or three-stranded antiparallel beta-sheets has become a field of intense interest. These studies have yielded great insights into the principles governing the stability and folding of beta-hairpins and antiparallel beta-sheets. This chapter reviews briefly those principles and describes a protocol for the de novo design of beta-sheet-forming peptides based on them. Criteria to select appropriate turn and strand residues and to avoid aggregation are provided. Because nuclear magnetic resonance is the most appropriate technique to check the success of new designs, the nuclear magnetic resonance parameters characteristic of beta-hairpins and three-stranded antiparallel beta-sheets are given.

Anabaena apoflavodoxin hydrogen exchange: On the stable exchange core of the α/β(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal α/β protein organized in three layers: a central β‐sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (kex < 10−1 min−1). According to their distribution in the structure, protein stability is highest on the β‐sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other α/β (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central β‐strands and in helix 5 is common to the cores. Proteins 2001;43:476–488.