Direct NMR observation of a substrate protein bound to the chaperonin GroEL

Abstract

The reaction cycle and the major structural states of the molecular chaperone GroEL and its cochaperone, GroES, are well characterized. In contrast, very little is known about the nonnative states of the substrate polypeptide acted on by the chaperonin machinery. In this study, we investigated the substrate protein human dihydrofolate reductase (hDHFR) while bound to GroEL or to a singlering analog, SR1, by NMR spectroscopy in solution under conditions where hDHFR was efficiently recovered as a folded, enzymatically active protein from the stable complexes upon addition of ATP and GroES. By using the NMR techniques of transverse relaxation-optimized spectroscopy (TROSY), crosscorrelated relaxation-induced polarization transfer (CRIPT), and cross-correlated relaxation-enhanced polarization transfer (CRINEPT), bound hDHFR could be observed directly. Measurements of the buildup of hDHFR NMR signals by different magnetization transfer mechanisms were used to characterize the dynamic properties of the NMR-observable parts of the bound substrate. The NMR data suggest that the bound state includes random coil conformations devoid of stable native-like tertiary contacts and that the bound hDHFR might best be described as a dynamic ensemble of randomly structured conformers.dihydrofolate reductase ͉ transverse relaxation-optimized spectroscopy (TROSY) ͉ cross-correlated relaxation-induced polarization transfer (CRIPT) ͉ protein folding

Coupling between allosteric transitions in GroEL and assisted folding of a substrate protein

“…Chains that do not bind to GroEL are most likely to have a native-like R g in the initial conformation. These results are in accord with recent NMR experiments (24) that showed that only random-coil states of dihydrofolate reductase are captured by GroEL.…”

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

“…To further characterize the NMR-observable polypeptide segments of rhodanese bound to SR1, we compared the efficiencies of 1 H magnetization transfer in backbone 15 25 . For rhodanese bound to SR1, T I % 3.5 ms and A C/I % 0.25 were obtained [ Fig.…”

“…1(D)], as compared to previously reported values of T I % 2.5 ms and A C/I % 0.3 for hDHFR in complex with SR1. 25 The longer T I value for rhodanese is due to slower 1 H N autorelaxation, which indicates that rhodanese retains more flexibility than hDHFR when bound to SR1. The A C/I values for both substrate proteins are small when compared to the value of A C/I % 0.8 predicted for 15 25 …”

“…Rhodanese is also a stringent substrate protein, that is, in contrast to hDHFR it is unable, in the absence of the chaperonin, to produce the native fold on dilution from denaturant at neutral pH and 22 C, which leads instead to nonspecific aggregation. 25,29 Because of the small chemical shift dispersion, the NMR signals are overlapped in clusters, so that it is difficult to estimate the number of residues observed in the spectrum of Figure 1(A). In the following sections, we used additional NMR experiments and biochemical studies of the rhodanese-SR1 complex to support a more detailed interpretation of the data in Figure 1(A) and to assess their biological relevancy.…”

NMR characterization of the interaction of GroEL with amyloid β as a model ligand

“…Hydrogen-deuterium exchange studies suggested that substrates lacked well-defined tertiary structure packing although the degrees of structural disorder were different for different proteins [7][8][9][10]. Direct NMR observations indicated that GroEL-bound substrate proteins were largely unstructured and highly dynamic [11,12]. However, arguments against the chaperoning mechanisms of GroEL are still controversial [13].…”