Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 AE 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 AE 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.protein folding | single-molecule FRET | coil-globule transition | polymer theory I t has become increasingly clear that the structure and dynamics of unfolded proteins are essential for understanding protein folding (1-3) and the functional properties of intrinsically disordered proteins (IDPs) (4-6). Theoretical concepts from polymer physics (7-9) have frequently been used to describe the properties of unfolded polypeptide chains (4, 10, 11) with the goal to establish the link between protein folding and collapse (12-15). However, the methodology to test many of these concepts experimentally has only become available rather recently (2,16,17). A considerable body of experimental and theoretical work suggests that the dimensions of unfolded proteins depend on parameters such as amino acid composition (4), temperature (18), and solvent quality (3,10,15,19). The continuous collapse of polymers has been treated exhaustively by a number of theories (20-24) based on general principles that relate the dimensions and the length of a chain to its free energy. However, a prerequisite for the quantitative application of these theories and their comparison to experimental results is that the dimensions of the Θ-state are known, which serves as an essential reference state. At the Θ-point*, chain-chain and chain-solvent interactions balance such that the polymer is at a critical point, at which the thermodynamic phase boundaries disappear. As a result, the polypeptide chain obeys the same length scaling as an ideal chain without excluded volume and intrachain interactions. However, the Θ-conditions for protein chains are unknown. Besides its importance for obtaining the corre...

The authors note that on page 4454, left column, 2nd full paragraph, lines 7-9, "For example, oxidation catalysts are able to reduce N 2 O emissions ∼70% compared with models without the technology (22)" should instead appear as "For example, advanced three-way catalysts are able to reduce N 2 O emissions ∼65% compared with models without the technology (22)."