Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.

Crowding and hydration effects on protein conformation: a study with sol-gel encapsulated proteins.

We are developing an experimental system for testing the effects of macromolecular crowding and molecular confinement on protein structure. In the present study, solvent effects on the secondary structure of two proteins were examined by circular dichroism following encapsulation in the hydrated pores of a silica glass matrix by the sol-gel method. Changes in the unfolded conformations of encapsulated apomyoglobin and reduced serum albumin were analyzed after equilibration with aqueous solutions of natural osmolytes, short-chain alcohols, polyethylene glycol, and a complete series of Hofmeister cations. In many instances, the alpha-helical content of the encapsulated protein was increased by addition of solutes at concentrations that have no effect on the protein in the absence of the glass. The results are discussed from the perspective of water structure. We argue that perturbed water at the silica interface causes an increase in the average free energy of the bulk water phase which, consequently, diminishes the strength of the hydrophobic effect inside the glass matrix and destabilizes the conformation of encapsulated proteins. We propose that solutes can increase the strength of the hydrophobic effect and influence folding equilibria without directly interacting with the protein. A hypothesis is provided for the apparent paradox that kosmotropic (strongly water binding) anions favor native protein structure, whereas chaotropic (weakly water binding) cations enhance native protein structure. The encapsulation results suggest that macromolecular crowding and molecular confinement are accompanied by hydration effects that may oppose or potentiate the stabilizing effects of excluded volume on protein structure, depending on the surface chemistry of the crowding agent and its influence on bulk water structure. In the crowded environment of a living cell, excluded volume effects, surface-induced water structure, and compatible solutes are expected to complement the dominant forces in protein folding.

Molecular confinement influences protein structure and enhances thermal protein stability.

The sol-gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, alpha-lactalbumin, and metmyoglobin retained native-like solution structures following sol-gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the alpha-helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.

Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant.

Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of familial amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably retains the gain-of-function activity that leads to disease. We demonstrate that Cu(2+) does not bind to the copper-specific catalytic site of H46R CuZnSOD and that Cu(2+) competes with other metals for the zinc binding site. Most importantly, Cu(2+) was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR spectroscopy, and chemical derivatization. Cysteine 111 was pinpointed as the position of the reactive ligand by tryptic digestion of the modified protein and by mutational analysis. This solvent-exposed residue may play a role in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine 111 residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal insertion during biosynthesis of wild-type CuZnSOD in vivo.