Molecular chaperones in protein folding and proteostasis

Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.     

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-? crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.     

Real-time observation of trigger factor function on translating ribosomes

The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.