RESUMEN
The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state. In the case of YY-DPETGT-WY, another submicrosecond folding species [Davis, C. M., Xiao, S., Raleigh, D. P., and Dyer, R. B. (2012) J. Am. Chem. Soc. 134, 14476-14482], a hydrophobic cluster provides the latter. In the case of HP7, the Coulombic interaction between the terminal NH3(+) and CO2(-) units provides this; a C-terminal Glu to amidated Ala mutation results in a 5-fold retardation of the folding rate. The effects of mutations within the reversing loop indicate the balance between loop flexibility (favoring fast conformational searching) and turn formation in the unfolded state is a major factor in determining the folding dynamics. The -NAAAKX- loops examined display no detectable turn formation propensity in other hairpin constructs but do result in stable analogues of HP7. Peptide KTW-NAAAKK-WTE displays the same fold stability as HP7, but both the folding and unfolding time constants are greater by a factor of 20.
Asunto(s)
Mutación , Fragmentos de Péptidos/química , Pliegue de Proteína , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Conformación ProteicaRESUMEN
Minimized beta hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, DeltaG(U)(298) >or= +7 kJ/mol) and 12-residue (KTWNPATGKWTE, DeltaG(U)(298) = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-pi interaction between Lys(1) and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hepsilon3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small DeltaC(p) for unfolding (+420 J K(-)(1)/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH(2)) with the fold stability reduced to DeltaG(U)(278) = -0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction.
Asunto(s)
Secuencias de Aminoácidos , Péptidos/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , TermodinámicaRESUMEN
Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin (k(F)(278)=1.5 x 10(4)/s). GB1 hairpin analogs spanning a wide range of thermodynamic stabilities (DeltaG(U)(298)=-3.09 to+3.25 kJ/mol) were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive side-chain/side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by "zipper" folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.
Asunto(s)
Mutación , Pliegue de Proteína , Espectroscopía de Resonancia MagnéticaRESUMEN
Cold denaturation is a general phenomenon in globular proteins, and the associated cold-denatured states of proteins have important fundamental and practical significance. Here, we have characterized the cold-denatured state of a beta-hairpin forming peptide, MrH3a, in 8% hexafluoro-2-propanol (HFIP) and the dynamics of its refolding following a laser-induced T-jump. Beta-hairpins constitute an important class of protein structural elements, yet their folding mechanisms are not fully understood. Characterization of MrH3a using NMR, CD, and IR spectroscopies reveals residual structure in the cold-denatured state, in contrast with the highly disordered heat-denatured state. The residual structure in the cold-denatured state comprises relatively compact and solvent protected conformations. Furthermore, we find a substantial acceleration in the rate of folding from the cold-denatured state compared to that of the heat-denatured state. In addition, the cold-denatured state is not populated in 20% HFIP; folding occurs only from the fully unfolded state and is significantly slower. We interpret the acceleration of the folding rate of MrH3a in 8% HFIP as a direct consequence of the collapsed conformations of the cold-denatured state. Finally, there may be some reduction of the loop search cost when starting from the cold-denatured state, since this state may have some of the stabilizing cross-strand interactions already formed.
Asunto(s)
Frío , Modelos Químicos , Péptidos/química , Pliegue de Proteína , Termodinámica , Dicroismo Circular , Calor , Humanos , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Propanoles/química , Desnaturalización Proteica , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A detailed analysis of peptide backbone amide (H(N)) and H alpha chemical shifts reveals a consistent pattern for beta hairpins and three-stranded beta sheets. The H alpha's at non-hydrogen-bonded strand positions are inwardly directed and shifted downfield approximately 1 ppm due largely to an anisotropy contribution from the cross-strand amide function. The secondary structure associated H alpha shift deviations for the H-bonded strand positions are also positive but much smaller (0.1-0.3 ppm) and the turn residues display negative H alpha chemical shift deviations (CSDs). The pattern of (H(N)) shift deviations is an even better indicator of both hairpin formation and register, with the cross-strand H-bonded sites shifted downfield (also by approximately 1 ppm) and with diagnostic values for the first turn residue and the first strand position following the turn. These empirical observations, initially made for [2:2]/[2:4]-type-I' and -II' hairpins, are rationalized and can be extended to the analysis of other turns, hairpin classes ([3:5], [4:4]/[4:6]), and three-stranded peptide beta-sheet models. The H alpha's at non-hydrogen-bonded sites and (H(N))'s in the intervening H-bonded sites provide the largest and most dependable measures of hairpin structuring and can be used for melting studies; however the intrinsic temperature dependence of (H(N)) shifts deviations needs to reflect the extent of solvent sequestration in the folded state. Several observations made in the course of this study provide insights into beta-sheet folding mechanisms: (1) The magnitude of the (H(N)) shifts suggests that the cross-strand H-bonds in peptide hairpins are as short as those in protein beta sheets. (2) Even L-Pro-Gly turns, which are frequently used in unfolded controls for hairpin peptides, can support hairpin populations in aqueous fluoroalcohol media. (3) The good correlation between hairpin population estimates from cross-strand H-bonded (H(N)) shift deviations, H alpha shift deviations, and structuring shifts at the turn locus implies that hairpin folding transitions approximate two-state behavior.