We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin ␣ suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.FRET ͉ polymer ͉ protein folding ͉ secondary structure ͉ chain dimensions T here is an increasing interest in the properties of unfolded proteins and their roles in the folding and cellular functions of proteins. A key motivation is that many proteins are marginally stable and only fold in the presence of their ligands or binding partners, opening new regulatory possibilities (1, 2). An important reason for recent progress is the growing availability of methods that provide structural information on these conformationally heterogeneous systems, such as NMR (3), scattering methods (4, 5), and single-molecule . Although NMR provides mostly local details, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and single-molecule FRET provide overall hydrodynamic or long-range distance information. An important advantage of single-molecule FRET is the separation of folded and unfolded subpopulations (9). As a result, unfolded state properties can be investigated even in the presence of folded molecules (i.e., under near-native conditions, which are physiologically most relevant). This advance has led to the observation of a continuous collapse of the unfolded state with decreasing denaturant concentrations (10), a behavior that now has been demonstrated for a large number of proteins (11-18) and peptides (19). Recent advances in the application of theoretical models have led to a quantitative description of this unfolded state collapse in terms of polymerphysical concepts (15,(20)(21)(22)(23). Such chain compaction also has been demonstrated to result in increased internal friction and a slowdown of intramolecular dynamics of the polypeptide (19, 26), which can affect...

Large oligomeric proteins are usually thought to fold and assemble hierarchically: Domains fold and coalesce to form the subunits, and folded subunits can then associate to form the multimeric structure. We have investigated the refolding pathway of the beta-sheet protein pea seed lectin using spectroscopic and hydrodynamic techniques. In vivo, it is proteolytically processed post-translationally, so that the single-domain subunits of the initial homodimer themselves become heterodimers of intertwined fragment polypeptide chains. Despite this complex topology, mature pea seed lectin reassembles with considerable efficiency at low total protein concentration (10 mug/mL) and low temperature (10 degrees C), albeit very slowly (t1/2 approximately 2 days). Contrary to expectations, the primary assembly product is not the intact beta-sheet domain, but the larger fragment chains first dimerize to form the native-like subunit interface. The smaller fragment chains then associate with this preformed dimer.