Biomedical subjects
Publications and source records attributed to Jörg Martin.
Coiled coils are versatile structural modules that engage in a variety of cellular activities. Recent studies illuminate their role as substrate-binding elements in the chaperone cofactor prefoldin and in the AAA+ ATPases involved in protein (un)folding processes. The use of coiled coils to mediate the binding of non-native proteins represents a novel strategy in chaperone design and a new function for coiled coils.
The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.
We report the characterization of the first chaperonin (Mm-cpn) from a mesophilic archaeon, Methanococcus maripaludis. The single gene was cloned from genomic DNA and expressed in Escherichia coli to produce a recombinant protein of 543 amino acids. In contrast with other known archaeal chaperonins, Mm-cpn is fully functional in all respects under physiological conditions of 37 degrees C. The complex has Mg(2+)-dependent ATPase activity and can prevent the aggregation of citrate synthase. It promotes a high-yield refolding of guanidinium-chloride-denatured rhodanese in a nucleotide-dependent manner. ATP binding is sufficient to effect folding, but ATP hydrolysis is not essential.
Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.
AAA proteins have entered the molecular realm after being known primarily for their wide range of different functions. Structural studies have highlighted the organization of their constituent ATPase domains and indicate that hexamerization in combination with unfoldase activity is a common underlying feature of this ubiquitous protein family.
Chaperonins assist in the acquisition of native protein structure in the cell by providing a shielded environment for a folding polypeptide chain, generated by the interior surface of their cylindrical structure. The folding chain is isolated from the highly crowded cytoplasm, but at the same time confined within the chaperonin folding cage. Both confinement and macromolecular crowding can affect folding kinetics and yields, the modus operandi of chaperonins and their interaction with their protegés. Recent experimental data, as well as computer simulations, provide increasing evidence that the particular physico-chemical conditions prevailing in the cellular interior have to be taken into account when trying to unravel the processes of cellular protein folding.