The interplay between secondary structure and hydrophobic collapse and how they define the molecular mechanisms by which proteins fold and unfold remains poorly understood. One reason is that to get physically meaningful information one must be able to inspect the distributions of stochastic behaviors of individual molecules and trap them on the act (i.e. resolve individual (un)folding transition paths). This is accessible to atomistic computer simulations, which offer a wealth of structural information with picosecond resolution, but use approximate molecular force-fields and thus must be validated with comparable experimental benchmarks. Experimentally, the problem can only be approached by single-molecule spectroscopic methods, but they have not had the time-resolution necessary to access the transition path timescales.<br/><br/>We are investigating protein folding at the single-molecule level combining advanced fluorescence and force spectroscopic methods. From the viewpoint of SM-fluorescence, we have developed a suite of approaches based on FRET detection that permit to push the envelope of single-molecule fluorescence detection of protein folding and enable the direct measurement of sub-millisecond (un)folding events as well as the molecular transition paths. On the force spectroscopy forefront, we have recently demonstrated the possibility of measuring the reversible folding-unfolding and transition paths of mechanically pliable fast-folding proteins (unfolding forces &lt;10 pN) using atomic force microscopy. Equipped with these complementary techniques we are investigating the folding-unfolding process of several fast folding proteins using chemical denaturing (by fluorescence) and mechanical (by force) perturbations. The idea behind using fast-folding proteins is that their relatively small free energy barriers (&lt;3 k<sub>B</sub>T) make their transition paths comparatively slow. An additional advantage is that fast-folding proteins fold onto the simplest types of structural folds, which facilitates extracting mechanistic and structural information from the limited resolution affordable to these experiments (end to end distance). To investigate the roles of secondary structure, fold topology, and amino acid sequence in protein folding we are studying four different fast-folding proteins, all of which consist of three secondary structure elements in an antiparallel arrangement.

Close look at the mechanisms of protein folding and unfolding via single molecule fluorescence and force spectroscopy methods

2019 Fall National Meeting

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