RESUMO
Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable ß-sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two ß-strands connected by varying loop lengths and composition (an example sequence: RWITVTI - loop - KKIRVWE). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RWITVTI-(GGGGKK)2 GGGG-KKIRVWE) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RWITVRIW-(GGGGKK)2 GGGG-WKTIRVWE) supports a remarkably stable ß-sheet (ΔGU = 6.3 kJ/mol at 295 K, Tm = â¼55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/síntese química , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Molecular simulation has been used to model the detailed folding properties of peptides, yet prospective computational peptide design by such approaches remains challenging and nontrivial. To test the accuracy of simulation-based hairpin design, we characterized the folding properties of a series of so-called ß-cap hairpin peptides designed to mimic a conserved hairpin of LapD, a bacterial intracellular signaling protein, both experimentally by NMR spectroscopy and computationally by implicit-solvent replica-exchange molecular dynamics using three different AMBER force fields (ff96, ff99sb-ildn, and ff99sb-ildn-NMR). A unique challenge presented by these designs is the presence of both a terminal Trp-Trp capping motif and a conserved GWxQ motif in the hairpin turn required for binding to LapG. Consistent with previous studies, we found AMBER ff96 to be the most accurate when used with the OBC GBSA implicit solvent model, despite its known bias toward ß-sheet conformations when used in explicit-solvent simulations. To gain microscopic insight into the folding landscape of the hairpin designs, we additionally performed parallel simulations on the Folding@home distributed computing platform using AMBER ff99sb-ildn-NMR with TIP3P explicit solvent. Markov state models (MSMs) built from trajectory data reveal a number of non-native interactions between Trp and other amino acid side chains, creating potential problems in achieving well-folded hairpin structures in solution.
Assuntos
Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência Conservada , Cinética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Solventes/químicaRESUMO
Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta-hairpin-like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram-negative, Escherichia coli but improved the activity against gram-positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram-negative versus gram-positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram-positive bacterium but less active against gram-negative bacterium. Our results show that there is not always a correlation between improved hairpin-structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Corynebacterium glutamicum/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estabilidade Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Beta sheets are inherently length-limited; adding residues to the ends of model ß-sheets does not necessarily grow the ß-sheet. Here, we present a method for extending ß-sheets to any length with a stabilizing repeat unit containing cross-strand Trp residues. Beta ribbons as long as 35â residues (approaching 100â Å in length) are reported and characterized.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
We have extended our studies of Trp/Trp to other Aryl/Aryl through-space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T-shape orientation. Designated as an edge-to-face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W â« Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225-232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197-200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp-cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross-strand aryl/aryl clusters for the design of stable ß-sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 337-356, 2016.
RESUMO
Building on our recent report of an active H2 production catalyst [Ni(PPh 2NProp-peptide)2]2+ (Prop = para-phenylpropionic acid, peptide (R10) = WIpPRWTGPR-NH2, p = D-proline and P2N = 1-aza-3,6-diphosphacycloheptane) that contains structured ß-hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction at longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.
RESUMO
Disulfide bonds between cysteine residues are essential to the structure and folding of many proteins. Yet their role in the design of structured peptides and proteins has frequently been limited to use as intrachain covalent staples that reinforce existing structure or induce knot-like conformations. In ß-hairpins, their placement at non-H-bonding positions across antiparallel strands has proven useful for achieving fully folded positive controls. Here we report a new class of designed ß-sheet peptide dimers with strand-central disulfides as a key element. We have found that the mere presence of a disulfide bond near the middle of a short peptide chain is sufficient to nucleate some antiparallel ß-sheet structure; addition of ß-capping units and other favorable cross-strand interactions yield hyperstable sheets. Strand-central cystines were found to be superior to the best designed reversing turns in terms of nucleating ß-sheet structure formation. We have explored the limitations and possibilities of this technique (the use of disulfides as sheet nucleators), and we provide a set of rules and rationales for the application and further design of disulfide-tethered "turnless" ß-sheets.
Assuntos
Dissulfetos/química , Peptídeos/química , Sequência de Aminoácidos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Protein dynamics on the microsecond (µs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent µs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior.
Assuntos
Álcool Desidrogenase/química , Espectrometria de Fluorescência/métodos , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Fluorescência , Geobacillus stearothermophilus/enzimologia , Histidina/química , Mutação , Conformação Proteica , Multimerização Proteica , Temperatura , Triptofano/químicaRESUMO
Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9-7.5 µs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.
Assuntos
Dobramento de Proteína , Espectroscopia de Ressonância Magnética , Mutação , Conformação ProteicaRESUMO
A hyperstable Pin1 WW domain has been circularly permuted via excision of the fold-nucleating turn; it still folds to form the native three-strand sheet and hydrophobic core features. Multiprobe folding dynamics studies of the normal and circularly permuted sequences, as well as their constituent hairpin fragments and comparable-length ß-strand-loop-ß-strand models, indicate 2-state folding for all topologies. N-terminal hairpin formation is the fold nucleating event for the wild-type sequence; the slower folding circular permutant has a more distributed folding transition state.
Assuntos
Peptídeos/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Catalytic, peptide-containing metal complexes with a well-defined peptide structure have the potential to enhance molecular catalysts through an enzyme-like outer coordination sphere. Here, we report the synthesis and characterization of an active, peptide-based metal complex built upon the well-characterized hydrogen production catalyst [Ni(P(Ph)2N(Ph))2](2+) (P(Ph)2N(Ph)=1,3,6-triphenyl-1-aza-3,6-diphosphacycloheptane). The incorporated peptide maintains its ß-hairpin structure when appended to the metal core, and the electrocatalytic activity of the peptide-based metal complex (≈100,000â s(-1)) is enhanced compared to the parent complex ([Ni(P(Ph)2N(APPA))2](2+); ≈50,500 s(-1)). The combination of an active molecular catalyst with a structured peptide provides a scaffold that permits the incorporation of features of an enzyme-like outer-coordination sphere necessary to create molecular electrocatalysts with enhanced functionality.
Assuntos
Complexos de Coordenação/química , Níquel/química , Peptídeos/química , Catálise , Técnicas Eletroquímicas , Hidrogênio/química , Hidrogenase/química , Modelos MolecularesRESUMO
An extensive series of covalently linked small molecule-peptide adducts based on a terminally capped-beta hairpin motif is reported. The constructs can be prepared by standard solid-phase Fmoc chemistry with one to four peptide chains linked to small molecule hubs bearing carboxylic acid moieties. The key feature of interest is the precise, buried environment of the small molecule, and its rigid orientation relative to one or more short but fully structured peptide chain(s). Most of this study employs a minimalist nine residue 'captide', a capped ß-turn, but we illustrate general applicability to peptides which can terminate in a beta strand. The non-peptide portion of these adducts can include nearly any molecule bearing one or more carboxylic acid groups. Fold-dependent rigidity sets this strategy apart from the currently available bioconjugation methods, which typically engender significant flexibility between peptide and tag. Applications to catalyst enhancement, drug design, higher-order assembly, and FRET calibration rulers are discussed.
Assuntos
Peptídeos/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Estrutura Secundária de ProteínaRESUMO
The present studies have shown that (13)C=O, (13)C(α) and (13)C(ß) of H-bonded strand residues in ß-hairpins provide additional probes for quantitating the extent of folding in ß-hairpins and other ß-sheet models. Large differences in the structuring shifts (CSDs) of these (13)C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three (13)C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from (13)C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the (13)C' and (13)C(ß) CSDs will provide the best differentiation with 100%-folded CSD values approaching -2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein ß-sheets. Our survey of reported (13)C shifts in ß-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Sequência de Aminoácidos , Aminoácidos/química , Isótopos de Carbono , Bases de Dados de Proteínas , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
Although much has been learned about the design of models of beta-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most beta-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, "unnatural" beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 + kJ/mol to beta-hairpin stability, surpassing all other interactions that stabilize beta-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel beta-strands and reduces fraying at the ends of hairpins and other beta-sheet models. Utilizing this motif, 10- to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The beta-cap can also be used to staple together short antiparallel beta-strands connected by a long flexible loop.
Assuntos
Proteínas/química , Acetilação , Sequência de Aminoácidos , Dicroísmo Circular , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismoRESUMO
The (13)C chemical shifts measured for designed ß-hairpins indicate that the structuring shifts (upfield for Cα and C', downfield for Cß) previously reported as diagnostic for ß-structuring in proteins appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of ß-strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for (13)C sites in ß-structures that should prove useful in the characterization of the folding equilibria of ß-sheet models.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Ligação de Hidrogênio , Estrutura Secundária de ProteínaRESUMO
By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i-2 Trp ring, a particularly stable (DeltaG(U) > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH(2), results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater beta-propensity of Trp. The pi-cation, and Trp/Gly-H(N) interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly-H(N) in -WX(n)G- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably because of its greater pi basicity.
Assuntos
Amidas/metabolismo , Cátions/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Triptofano/metabolismo , Amidas/química , Cátions/química , Ligação de Hidrogênio , Indóis/química , Indóis/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Peptídeos/genética , Mutação Puntual , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Termodinâmica , Temperatura de Transição , Triptofano/químicaRESUMO
Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH2, (a) bifurcated H-bonding involving the threonine amide H(N) and side chain OH and the N-terminal acetyl carbonyl and (b) an H-bond between the entgegen-H(N) of the C-terminal amide and the indole ring of Trp6 that stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6, has afforded < or = 10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH3CH2CO-WI pGXWTGPS (p = D-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298 K (97.5% at 280 K) with melting temperatures > 75 degrees C. The fold appears to display minimal fluxionality; a well-converged NMR structure rationalizes all of the large structuring shifts observed, and we suggest that these designed constructs can be viewed as microproteins.
Assuntos
Oligopeptídeos/química , Dobramento de Proteína , Água/química , Deutério , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Relação Estrutura-Atividade , TemperaturaRESUMO
Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a ß-hairpin conformation stabilized by cross-strand disulfides across the associated ß-strands. Here, we show that the disulfides are not essential. Other structuring means such as better ß-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless ß-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on AMPs. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active ß-hairpin peptides.