Brandon L. Kier

Stabilizing capping motif for β-hairpins and sheets

Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most β-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 β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands and reduces fraying at the ends of hairpins and other β-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 β-cap can also be used to staple together short antiparallel β-strands connected by a long flexible loop.

Very short peptides with stable folds: Building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone–amide interaction geometries

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 (ΔGU > 7 kJ/mol) truncated hairpin, Ac‐WI‐(D‐Pro‐D‐Asn)‐KWTG‐NH2, 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 β‐propensity of Trp. The π–cation, and Trp/Gly‐HN interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly‐HN in ‐WXnG‐ 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 π basicity. Proteins 2009.

Probing the Lower Size Limit for Protein-Like Fold Stability: Ten-Residue Microproteins With Specific, Rigid Structures in Water

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.

Enzyme design from the bottom up: an active nickel electrocatalyst with a structured peptide outer coordination sphere.

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 β-hairpin structure when appended to the metal core, and the electrocatalytic activity of the peptide-based metal complex (≈100,000 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.

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

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.

Fluorescence of tryptophan in designed hairpin and Trp-cage miniproteins: measurements of fluorescence yields and calculations by quantum mechanical molecular dynamics simulations.

The quantum yield of tryptophan (Trp) fluorescence was measured in 30 designed miniproteins (17 β-hairpins and 13 Trp-cage peptides), each containing a single Trp residue. Measurements were made in D(2)O and H(2)O to distinguish between fluorescence quenching mechanisms involving electron and proton transfer in the hairpin peptides, and at two temperatures to check for effects of partial unfolding of the Trp-cage peptides. The extent of folding of all the peptides also was measured by NMR. The fluorescence yields ranged from 0.01 in some of the Trp-cage peptides to 0.27 in some hairpins. Fluorescence quenching was found to occur by electron transfer from the excited indole ring of the Trp to a backbone amide group or the protonated side chain of a nearby histidine, glutamate, aspartate, tyrosine, or cysteine residue. Ionized tyrosine side chains quenched strongly by resonance energy transfer or electron transfer to the excited indole ring. Hybrid classical/quantum mechanical molecular dynamics simulations were performed by a method that optimized induced electric dipoles separately for the ground and excited states in multiple π-π* and charge-transfer (CT) excitations. Twenty 0.5 ns trajectories in the tryptophans lowest excited singlet π-π* state were run for each peptide, beginning by projections from trajectories in the ground state. Fluorescence quenching was correlated with the availability of a CT or exciton state that was strongly coupled to the π-π* state and that matched or fell below the π-π* state in energy. The fluorescence yields predicted by summing the calculated rates of charge and energy transfer are in good accord with the measured yields.

Captides: rigid junctions between beta sheets and small molecules

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 β‐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. Copyright

An improved capping unit for stabilizing the ends of associated β‐strands

Understanding protein beta structures has been hindered by the challenge of designing small, well‐folded β‐sheet systems. A β‐capping motif was previously designed to help solve this problem, but not without limitations, as the termini of this β‐cap were not fully available for chain extension. Combining Coulombic side chain attractions with a Trp/Trp edge‐to‐face interaction we produced a new capping motif that provided greater β‐sheet stability. This stability was maintained even in systems lacking a turn locus with a high propensity for chain direction reversal. The Coulombic cap was shown to improve β‐sheet stability in a number of difficult systems, hence providing an additional tool for protein structure and folding studies.

Circular permutation of a WW domain: folding still occurs after excising the turn of the folding-nucleating hairpin.

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 β-strand-loop-β-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.

Aryl‐Aryl interactions in designed peptide folds: Spectroscopic characteristics and placement issues for optimal structure stabilization

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 ≫ 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 β‐sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts.