15 September 1995 Transient laser spectroscopy of protein folding: detection and characterization of slow annealing processes
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Abstract
The unique structure of a protein is encoded in its characteristic sequence of amino acids; the processes by which this linear sequence collapses into a unique 3D structure remains an unsolved problem that represents one of the most challenging issues in fundamental biomolecular science. This so-called protein folding problem is the second half of the genetic code. Studies of this biological problem are complicated by the need to study dynamic behavior involving small populations of transient species in a solution environment. However, the use of advanced transient laser spectroscopy techniques based on intrinsic chromophores provides a powerful means to study this problem. Specifically, time-resolved phosphorescence of tryptophan (Trp) provides a means to study the dynamics associated with different regions of the protein surrounding the emitting Trp residue. Using these methodologies, we are able to study, in real time, the later stages of unfolding and refolding of the bacterial protein alkaline phosphatase, a nonspecific monoesterase. Results show the presence of several intermediate states, including states with significantly altered core structure that still exhibit complete biological activity. Moreover, the refolding of alkaline phosphatase following denaturation in either chaotropic denaturants or low pH reveals a relatively fast refolding leading to the biologically active state, while laser spectroscopy measurements show a soft core which is annealing to the native-like state on a time-scale long compared to the return of activity. The active refolded protein is also initially characterized by an increase in susceptibility to denaturant. The slow annealing of the core is consistent with the presence of high energy barriers that separate fully active, long-lived, kinetic intermediate states along the folding pathway, a description suggested in the rugged energy landscape model.
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Vinod Subramaniam, Nils Bergenhem, Ari Gafni, Duncan G. Steel, "Transient laser spectroscopy of protein folding: detection and characterization of slow annealing processes", Proc. SPIE 2524, National Science Foundation (NSF) Forum on Optical Science and Engineering, (15 September 1995); doi: 10.1117/12.219565; https://doi.org/10.1117/12.219565
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