Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE. Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.

How chaperones fold proteins.

Chaperones are a functionally related group of proteins assisting protein folding in the cell under physiological and stress conditions. They share the ability to recognize and bind nonnative proteins thus preventing unspecific aggregation. The underlying functional principles of the different chaperone classes are beginning to be understood. A landmark feature of molecular chaperones is the involvement of energy-dependent reactions in the folding process. Nucleotide binding to ATP-dependent chaperones (e.g. GroEL, Hsp70, Hsp90) leads to sometimes large conformational changes in the chaperone which allow to shift between high- and low-affinity states for substrate proteins. Interestingly, the ATPase activity which is the key determinant for functional cycles is tightly regulated by a set of co-chaperones. While for ATP-dependent chaperones binding sites for nucleotide and protein are found in one protein, in the case of ATP-independent chaperones (e. g. sHsps, SecB) the energy-dependent step is performed by another chaperone (Hsp70, SecA). Therefore, the ATP-independent chaperones can be regarded as efficient 'holding' components. Cooperation of different chaperone machineries creates a synergistic network of folding helpers in the cell, which allows to maintain protein homeostasis under conditions nonpermissive for spontaneous folding.

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.

Mutations that stabilize folding intermediates of phage P22 tailspike protein: folding in vivo and in vitro, stability, and structural context.

The folding of the trimeric phage P22 tailspike protein is affected by single amino acid substitutions designated temperature-sensitive folding (tsf) mutations. Their phenotypes are alleviated by two repeatedly isolated global suppressor (su) mutations (su V331A and su A334V) and by two additional substitutions (su V331G and su A334I), accessible through site-directed mutagenesis. We investigated the influence of the suppressor mutations on tailspike refolding in vitro, on its maturation at high expression levels in vivo, and on the rates of thermal unfolding of the native protein. All su mutations improved the folding efficiency in vitro and in vivo, but the relative effects of substitutions at position 334 were more pronounced in vivo, whereas the 331 substitutions were more effective in vitro. V331G caused the strongest increase in refolding yields of any single mutation, and was as effective as the V331A/A334V double mutation, where the two single mutations exhibited an additive effect. Both V331A and V331G retarded thermal denaturation, while A334V did not affect, and A334I accelerated unfolding. A334I is the first mutation found to affect the folding of the tailspike and the thermal stability of the native protein in opposite directions. The observed effects can be rationalized on the basis of the recently determined crystal structure of an N-terminally shortened tailspike. As the backbone dihedral angles of Val331 (phi = -119 degrees, psi = -142 degrees) are unusual for non-glycine residues, V331G and V331A may remove steric strain and thereby stabilize folding intermediates and the native protein. The beta-branched side-chains of Val and Ile substituted for Ala334 in the interior of the protein may improve a hydrophobic stack of residues in the large parallel beta-helix. This is likely important in loosely structured early folding intermediates, but not in the very rigid native structure, where the side-chain of Ile can hardly be accommodated.