Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering

Abstract

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.The stability and folding speed of a protein depend on the structures of the denatured as well as the native state, raising the question: do proteins fold more rapidly from a denatured state of expanded structures or from one of compact structures? Lattice simulations of simplified representations of proteins suggest that slow folding amino acid sequences collapse to compact structures with non-native topologies before folding, while fast folders collapse and fold simultaneously (1-4). We have begun to address this question experimentally with submillisecond small-angle x-ray scattering (SAXS) using synchrotron radiation and a microfabricated diffusional mixer to rapidly initiate folding. SAXS yields the radius of gyration, the most unambiguous measure of compactness. Here we show that, in contrast to the simulations, one of the fastest-folding proteins (cytochrome c: folding ϭ 400 s) first collapses to compact structures before forming the final native state. X-ray scattering by proteins in solution is sensitive to spatial variations in electron density. Scattering at the smallest angles yields the radius of gyration, R g , which in conjunction with the protein molecular weight provides a measure of the compactness of globular proteins. Additional structural information can be obtained from scattering at larger angles, which reflects electron density correlations on length scales shorter than R g . For a compact polymer, such as the native protein or compact denatured structures, I(q)q 2 increases at low q, goes through a maximum, and decreases at large q where I(q)␣q Ϫ4 [I(q) is the scattered intensity; q ϭ 4sin ͞, is the momentum transfer;is the x-ray wavelength, 1.54 Å; and 2 is the scattering angle] (5). In contrast, for a polymer chain undergoing a random walk in space, as can occur for an unfolded protein under strongly denaturing conditions, I(q)q 2 first increases, then plateaus (6), and, at large q, where I(q)␣q

Dynamics, Energetics, and Structure in Protein Folding

“…the roughness of the landscape) might be quite large (4). A dramatic demonstration of this effect is seen in the rate of collapse of unfolded cytochrome c, in which the strong non-specific interactions with the heme slow down the collapse rate to hundreds of microseconds (47)(48)(49)(50). The experiments on the fast-folding mutants of λ repressor (see previous section) suggest that the effective diffusion coefficient for folding of this protein is ~1/(2 microseconds) at 340 K (35).…”

Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

“…Although the precision of the data are poor and an attempt to fit it to a model will be at best questionable, we might be observing a sigmoidal transition in mean E. This transition is centered at about 4.4 M denaturant and could indicate an increase in protein dimensions with increase in denaturant concentration. Such a ''swelling'' of the denatured protein can occur because of a reduction in persistent specific structure or an increase in the ''solubility'' of the protein random coil (1,(31)(32)(33). It is noted that this shift occurs over a limited range of denaturant concentration (4 M to 5 M), close to the major unfolding transition region at 4 M denaturant (see the denaturation curves in Fig.…”

Microfluidic Assisted Self‐Assembly of pH‐Sensitive Low‐Molecular Weight Hydrogelators Close to the Minimum Gelation Concentration

“…[14] Microfluidic flow reactors can be potentially employed to control the pH medium at the nanoscale by controlling the flow of different pH solutions and different concentrations of the LMWG solution. Microfluidic platforms allowing mixing have been proved to be excellent tools to study protein denaturation, [15] protein fibrillation, [16] RNA refolding. [17] Microfluidic flow reactors could thus be used as a substitute to chemical species to modify the pH at the nanoscale in a controlled fashion avoiding heterogeneous detrimental competing effects during the addition of strong acids.…”