Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Abstract

We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

Is there an en route folding intermediate for cold shock proteins?

“…The reconfiguration time of Csp in the unfolded state in our simulations is on the order of 10 4 time units ($100 ns, which is comparable with experimental value, 20,59 50 ns). The work by Schuler and coworkers 21 suggested that around 20% of the b strands are formed after 1.3 ms dead time. This finding may be owing to the contribution of partially folded b1-b2-b3 strand and may be influenced by a contribution from a small fraction of the folded state.…”

“…With the advances in single-molecule techniques, valuable insights have been gained from recent single molecule fluorescence energy transfer (FRET) studies of the Csp from T. maritima. [17][18][19][20][21] The two-state folding dynamics has been demonstrated clearly at the single-molecule level. 17 In many experimental studies, 3,4,12 the fluorescence of a single tryptophan is used alone to probe the folding dynamics.…”

“…11 Such a fast collapse process was suggested mostly owing to nonspecific interactions 10 and involves a collapsed structure that is essentially unfolded except some structural elements that are similar to the folded state. Furthermore, the single-molecule study by Schuler and coworkers 21 suggested that around 20% of b sheets are formed in the fast collapse phase occurring in a dead time around 1.3 ms (the folding time under such conditions is around 19 ms). Single-molecule FRET experiments 21,65 showed, not surprisingly, that the unfolded states at lower concentration of denaturant are more compact than at higher concentration of denaturant.…”

“…Furthermore, the single-molecule study by Schuler and coworkers 21 suggested that around 20% of b sheets are formed in the fast collapse phase occurring in a dead time around 1.3 ms (the folding time under such conditions is around 19 ms). Single-molecule FRET experiments 21,65 showed, not surprisingly, that the unfolded states at lower concentration of denaturant are more compact than at higher concentration of denaturant. Intuitively, when Csp is switched from a high concentration of denaturant to a low concentration of denaturant, the former unfolded state will collapse ultrafast and relax to the new unfolded state at this new lower concentration of denaturant.…”

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

“…However it is not yet able to determine dissociation constants for proteins, measure the conformation and stability of proteins, or detect and quantify the interaction of a protein with small drug-like molecules or ligands. Greater sensitivity at the single molecule level has been achieved recently for the measurement of conformational changes by fluorescence correlation spectroscopy, though this again relies upon the labeling of proteins with a strong fluorophore such as Alexa or AttoOxa11 dyes, 24,25 or the use of highly fluorescent proteins such as yellow fluorescent protein (YFP). 26 Here we present a microfluidic measurement technique that enables the thermodynamic stability of reversibly unfolding proteins, and the affinity of a small molecule drug compound for a target protein, to be determined using small samples and with high precision.…”