Primary Peptide Folding Dynamics Observed with Ultrafast Temperature Jump

Abstract

The folding dynamics of proteins and polypeptides is a complex process involving different time and length scales. [1][2][3][4] Among the secondary structural elements the α-helix is the most commonly found configuration with its stability resulting from the unique hydrogen bonding; the C=O group of an amino acid at the position i forms a hydrogen bond with the N-H group of another amino acid located at the position i+4. The thermodynamic properties of the α-helix are understood in the context of the helix-coil transition, but the dynamics, being of many steps, have a whole range of time scales. The rates have been reported using a variety of experimental methods [for recent reviews see refs. 5-7] including absorption, NMR, Raman, infrared, and circular dichroism. The helix-coil transition was believed to occur on the microsecond timescale, and only recently, by means of fluorescence detection, were the rates measured to be as short as 300 ns, [8][9][10] prompting the association of the 300 ns results with "ultrafast" dynamics.[9] Theoretical models of helix-coil (polymer type) transitions and molecular dynamics (MD) simulations (see below) have also provided a range of time scales. For example, Schwarz,[11] using Zimm-Bragg nucleation and elongation parameters,[12] estimated the "relaxation time" to be 0.1 microsecond, whereas the time scale in MD simulations of folding (sub-ns to μs) depends on the length and sequence. In order to resolve the primary processes of folding, the dynamics have to be observed with the shortest time resolution possible.In this contribution, we report the ultrafast folding dynamics of the α-helix with time resolution three orders of magnitude shorter than previously reported with fluorescence detection. The in situ measured heating time (3.5 ps), with the ultrafast T-jump method, [13] is determined by the water relaxation time of the peptide in pure water, as described below. Earlier, a T-jump with time resolution of 70 ps was used to study proteins by heating through dye molecules in the solution. [14] Here, the heating is directly through the water vibrations on the 3.5 ps time scale. We studied the α-helical alanine-based pentapeptide, Ac-W-(A) 3 -H + -NH 2 (Wh5), in an acetate buffer at pH = 4.8. Other alanine-based peptides with

Dominance of misfolded intermediates in the dynamics of α-helix folding

“…Indeed, helix folding experiments demonstrated that the timescale of the nucleation step could be relatively fast: α-helix nucleation, when observed under no-zipping conditions, occurs on a few-nanosecond timescale, which is consistent with analytical and computational assessments carried out in this (8,9) and other laboratories (28,29). In contrast, the overall helix folding timescale at room temperature is broadly distributed depending on sequence, typically being hundreds of nanoseconds (18,19).…”

“…Thus, relatively little was known about the associated kinetics until the end of the 1990s (3). With the advent of temperature-jump (T-jump) experimental techniques, the rates of the helix−coil transitions became readily measurable (4-6), whereas the progress in temporal resolution spanning both the nanosecond (7) and picosecond (8) regimes elucidated the early steps of folding/unfolding. Importantly, studies of the elementary steps accompanying such processes can now uncover the nature of kinetic-intermediate states, as well as their characteristic lifetimes (8,9).…”

N-Protonated Isomers as Gateways to Peptide Ion Fragmentation

“…Thus, a conformational change is required to mobilize the proton from the N-terminus to the C-terminal amide nitrogen. It is known that globule-helix conformational transitions of peptides are rapid [38,39]. Seemingly at odds with our conclusion, experimental and molecular dynamics (MD) studies have suggested that no helical structures should be expected from protonated Ala 15 H + [40].…”

Nanomechanics and intermolecular forces of amyloid revealed by four-dimensional electron microscopy

“…We note that the rise of the expansion is within the T-jump resolution. This rise simply reflects the time for heat transfer from the dye to the protein and it is typically on a subnanosecond timescale (16). Here, it is of no relevance.…”