Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL.

Nuclear magnetic resonance (NMR) observation of the uniformly (2) H,(15) N-labeled stringent 33-kDa substrate protein rhodanese in a productive complex with the uniformly (14) N-labeled 400 kDa single-ring version of the E. coli chaperonin GroEL, SR1, was achieved with the use of transverse relaxation-optimized spectroscopy, cross-correlated relaxation-induced polarization transfer, and cross-correlated relaxation-enhanced polarization transfer. To characterize the NMR-observable parts of the bound rhodanese, coherence buildup rates by different magnetization transfer mechanisms were measured, and effects of covalent crosslinking of the rhodanese to the apical binding surface of SR1 were investigated. The results indicate that the NMR-observable parts of the SR1-bound rhodanese are involved in intracomplex rate processes, which are not related to binding and release of the substrate protein from the SR1 binding surface. Rather, they correspond to mobility of the stably bound substrate, which thus appears to include flexibly disordered polypeptide segments devoid of long-lived secondary structures or tertiary folds, as was previously observed also with the smaller substrate human dihydrofolate reductase.