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The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

The role of beta-turns in dictating the structure of a beta-barrel protein is assessed by probing the tolerance of the central beta-turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild-type Pro47-Ser48-Gly49-Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure. Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II beta-turn disrupt the native structure severely. Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments. These mutant proteins do not bind copper. Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed. These results indicate that the central beta-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the beta-barrel structure. These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins.

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.