The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods

Abstract

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, R, of 1.6 sec in 3 M guanidine-HCl at pH 7 and 20°C. The value of R increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, R is 2.5 sec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding. Little is known, however, about the dynamics between different conformers in the unfolded state. Probing these conformational transitions is not trivial for two reasons. First, the dynamics are expected to be rapid (sec) and inaccessible to NMR and other commonly used experimental techniques. Second, the difference in spectroscopic signatures between two conformational states is small or even absent, thus making it difficult to find a suitable probe. In this study, we have used fluorescence self-quenching of tetramethyl rhodamine (TMR) as analyzed by fluorescence correlation spectroscopy (FCS) to study the dynamics of the unfolded state under different solution conditions.To perform these experiments we have incorporated a fluorescent probe in two positions of the intestinal fatty acid binding protein (IFABP), 48 residues apart, and have directly measured both the rate of diffusion of the whole molecule and the rate of internal dynamics of the chain in the unfolded state. IFABP consists of two ␤-sheets enclosing a large cavity into which the fatty acid binds (3). The protein (131 residues, 15 kDa) does not contain either cysteine or proline residues, but numerous mutations of residues to cysteine, to which fluorescence probes have been covalently attached (4) have been made with few deleterious effects. Thus, this protein provides an excellent model system for these studies. The persistence of structure in the unfolded state of IFABP was observed by Hodsdon and Frieden (5). In the present study two polar residues, in the two different ␤-sheets, have been mutated to cysteine (D59C͞E107C) and then modified with TMR-5-maleimide. Two other mutants were generated, each with only one residue replaced by cysteine and modified by TMR (D59C-TMR and E107C-TMR). Relative to the labeled single mutants, the doubly labeled mutant is significantly quenched in the presence of 2 M guanidine-HCl (Gdm⅐HCl). We conclude that this quenching (fluorescence self-quenching) ...

Coil–globule transition in the denatured state of a small protein

“…Recent experiments (22,23) suggest that the end-to-end distance dynamics for a denatured protein of the size of protein L are on the microsecond time scale, whereas the smFRET data are averaged over millisecond fluorescence bursts. We therefore took the mean E of the denatured-state distribution as an average over the end-to-end distance distribution, P(R ee ), of the protein…”

Managing Free-energy Barriers in Nuclear Pore Transport

“…Equation (1) can be evaluated from first principles assuming that the dynamics of l is slow compared to the characteristic time scales of the solvent molecules and the components, which constitute the barrier [15]. This condition is fulfilled since the radial relaxation time for random coiled polypeptides is in the μsec range [16] while the passive diffusion of macromolecules over length scales of the NPC is a msec phenomenon. Such an approach shows that F(l) is the free energy of the total system constrained to a particular value of l. D is the diffusion constant of the cargo-carrier complex.…”

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

“…In completely unfolded proteins at high denaturant concentration, diffusive interconversion of different conformations have been shown to occur on the Ϸ1-to Ϸ200-s time scale (54,55). The Ϸ1-s motions, which scale as n Ϫ3/2 , represent the time scale for the diffusive formation of a single long-range contact between distal regions of the protein chain separated by n residues (56).…”