Exploring protein-cofactor interactions on a molecular level is one of the major challenges in modern biophysics. Based on structural data alone it is rarely possible to identify how subtle interactions between a protein and its cofactor modulate the protein's reactivity. In the case of enzymatic processes in which paramagnetic molecules play a certain role, EPR and related methods such as ENDOR are suitable techniques to unravel such important details. In this contribution, we describe how cryogenic-temperature ENDOR spectroscopy can be applied to various LOV domains, the blue-light sensing domains of phototropin photoreceptors, to gain information on the direct vicinity of the flavin mononucleotide (FMN) cofactor by analyzing the temperature dependence of methyl-group rotation attached to C(8) of the FMN's isoalloxazine ring. More specifically, mutational studies of three amino acids surrounding the methyl group led to the identification of Asn425 as an important amino acid that critically influences the dark-state recovery of Avena sativa LOV2 domains. Consequently, it is possible to probe protein-cofactor interactions on a sub-angstrom level by following the temperature dependencies of hyperfine couplings.
Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.