α-Helix folding in the presence of structural constraints

Abstract

We have investigated the site-specific folding kinetics of a photoswitchable cross-linked ␣-helical peptide by using single 13 C ‫؍‬ 18 O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6°C), whereas at high temperatures (45°C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained ␣-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an ␣-helical segment within a protein, rather than an isolated ␣-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.cooperativity ͉ infrared spectroscopy ͉ molecular dynamics simulation ͉ peptide folding

Effect of viscogens on the kinetic response of a photoperturbed allosteric protein

“…32. To force the photoswitch to be either in the cis or the trans-configuration, the double minimum potential for the central C-N=N-C dihedral angle was replaced by a single minimum potential, [33][34][35] and the force constant increased by a factor 3 such that the potential around the minimum agrees reasonably well with that of the original double minimum potential.…”

“…43,44 Also the folding of protein secondary structure motifs of small peptides such as α-helix folding, which are known to deviate from two-state behavior, commonly follows stretched exponential kinetics with stretching factors around β = 0.7-0.8. 34,45 At the same time, the viscosity de- pendence of such processes has been shown to be fractional with γ = 0.6. 7 Based on MD simulations, Ref.…”

Effects of side chains in helix nucleation differ from helix propagation

“…Disulfide-linked HBS helices can be derived from oxidation of the parent bisthiol peptides allowing a facile approach to control the conformation of the peptide (31)(32)(33)(34). We based our initial peptide design on a short segment from the p53 activation domain: XQEG*FSDLWKLLS (1), where X and G* represent the dsHBS-constrained residues (35).…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…NMR spectroscopy can elucidate dynamics on many timescales, even down into the picosecond regime, but it does so via relaxation experiments, which works only for equilibrium fluctuations. With "direct time resolution", we have in mind to study non-equilibrium processes in a pump-probe fashion, such as the triggered (un)folding of a protein [4][5][6][7][8][9][10][11] or the propagation of a perturbation through an allosteric system [12][13][14]. The direct time resolution of a spectroscopic method is dictated by the dephasing time of its transitions, which is a few picoseconds for typical vibrational bands in the solution phase, in contrast to millisecond dephasing of NMR.…”

“…The protein should have only one of these amino acids at the desired position, and in this regard, Met is advantageous as it is relatively rare in natural proteins, whereas His is often crucial for their function. Note that we have disregarded cysteine as another possible anchor point for a label [29], since we want to keep that amino acid free for adding other functionalities, such as a photoswitch [6,8,12,13,59] used to initiate a non-equilibrium process (see the example highlighted below). We reiterate that being able to initiate a non-equilibrium process is a prerequisite to extend the accessible time window beyond the vibrational lifetime of the label.…”