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

Abstract

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.

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…The conformations of the proteins human DHFR [224] and rhodanese [225] within the GroEL chaperonin were analyzed by 2D [ 15 N, 1 H]-TROSY, showing resonances in the random coil region, indicating a lack of regular secondary and tertiary structure. The observed line-widths were broad, reflecting either slow tumbling of the large complex, internal motion of the bound substrate, the presence of the substrate in multiple bound conformations, or some combination of these factors.…”

Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR

“…Directly observed 1 H-15 N correlation methods were suggestive (but not conclusive) of protein substrates being disordered and dynamic while bound to GroEL, because all observable cross-peaks had chemical shifts characteristic of random coil and significant portions of the bound proteins were NMR-invisible because the cross-peaks for every residue type were underrepresented (16,17). This conclusion, which was essentially one of exclusion because the portion of the protein substrates directly bound to GroEL could not be observed, is confirmed by the current work, which directly probes the NMR-invisible dark state of the bound substrate at every residue position.…”

“…Conversely, hydrogen-deuterium exchange experiments (14,15) imply that the secondary structure is sufficiently destabilized or disrupted upon binding to GroEL to allow substantial backbone amide hydrogen-deuterium exchange to occur. Likewise, 1 H-15 N correlation experiments, designed specifically for large (>>100 kDa) protein assemblies, suggest that bound protein substrates are dynamic and largely unfolded, because the few cross-peaks observed are located at characteristic random coil positions (16,17).…”

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

“…Yet another interesting spin is studying structures of GroEL/GroELS by a variety of techniques [4][5][6]15] and applying these approaches to studying protein and peptide structures in a bound state with GroEL by X-ray, NMR and cryo-electron microscopy [16][17][18][19][20].…”