Clara M. Santiveri

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.

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.

Interaction networks in protein folding via atomic-resolution experiments and long-time-scale molecular dynamics simulations

The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue–residue couplings associated with cooperative folding emerges. Such thermodynamic residue–residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process. Our results on gpW indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins.

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.

A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF–VEGFR interaction

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.