Molecular analysis of changes in DNA binding affinity and bending extent induced by the mutations on the aromatic amino residues in Cren7.

Proteins from Crenarchaeal organisms exhibit remarkable thermal stability. The aromatic amino acids in Cren7, a Crenarchaeal protein, regulate protein stability and further modulate DNA binding and its compaction. Specific aromatic amino acids were mutated, and using spectroscopic and theoretical approaches, we have examined the structure, DNA binding affinity, and DNA bending ability of mutants. and compared with wild-type (WT) Cren7. The reverse titration profiles were analysed by a noncooperativeMcGhee-von Hippel model to estimate affinity constant (Ka) and site size (n) associated with binding to the DNA. Biolayer interferometry (BLI) measurements showed that the binding affinity decreased at higher salt concentrations. For theoretical analysis of extent of DNA bending, radius of gyration and bending angle were compared for WT and mutants. Time evolution of order parameters based on translational and rotational motion of tryptophan residue (W26) was used for qualitative detection of stacking interactions between W26 of Cren7 and DNA nucleobases. It was observed that orientation of W26 in F41A favored formation of a new lone pair-lone pair interaction between DNA and Cren7. Consequently, in thermostable proteins, the aromatic residues at the terminus maintain structural stability, whereas the residues at the core optimize the degree of DNA bending and compaction.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and ß-subdomain. We identified a flexible region within the rigid ß-subdomain that gives way under load, thus opening up the α/ß interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Knotting and unknotting of a protein in single molecule experiments.

Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.