Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function.

Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast.

Posttranslational modification and conformational state of heat shock protein 90 differentially affect binding of chemically diverse small molecule inhibitors.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences.

Enforced N-domain proximity stimulates Hsp90 ATPase activity and is compatible with function in vivo.

Hsp90 populates distinct open and closed conformations mediated by transient N-terminal dimerization. To investigate the mechanistic role of these large conformational changes, we designed Hsp90 with an N-terminal coiled-coil to clamp the termini together and enforce N-domain proximity. Biophysical analyses demonstrate that the coiled-coil effectively maintains N-domain proximity in the absence of ATP, a condition that favors the open state of Hsp90.

A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states.