TEMPERATURE-DEPENDENCE OF BACKBONE DYNAMICS IN LOOPS OF HUMAN MITOCHONDRIAL HEAT-SHOCK-PROTEIN-10

A highly flexible, yet conserved polypeptide loop of Hsp10 mediates bi nding to Hsp60 in the course of chaperonin-dependent protein folding. Previous transferred nuclear Overhauser effect (trNOE) studies with pe ptides based on the mobile loop of the Escherichia coli and bacterioph age T4 Hsp10s suggested that the mobile loop adopts a characteristic h airpin turn upon binding to the E. coli Hsp60 GroEL. In this paper, we identify the sequence and characterize the nascent structure and dyna mics of the 18-residue mobile loop in the N-15-enriched human Hsp10. W e also identify four residues of another flexible loop, the roof beta hairpin. The mobile loop and/or roof beta hairpin of several subunits are absent from the X-ray crystal structure of human Hsp10. NMR data s uggest that the mobile loop of Hsp10 preferentially samples a hairpin conformation despite the fact that the backbone motion resembles that of a disordered polypeptide. Analysis of backbone dynamics by measurem ent of N-15 relaxation times, T-1 and T-2, and the H-1-N-15 nuclear Ov erhauser effect (H-1-N-15 NOE) indicates that motion is greatest near the center of the loop. Inversion of the temperature dependence of the T-1 near the center of the loop marks a transition to motion with a d ominant time scale of less than 3 ns. Analysis of the relaxation data by spectral density mapping shows that subnanosecond motion increases uniformly along the loop at elevated temperatures, whereas nanosecond motion increases near the ends of the loop and decreases near the cent er of the mobile loop. The transition to dominance by fast motion in t he center of the loop occurs at a distance from the well-structured pa rt of Hsp10 that is equal to the persistence length of an unstructured polypeptide. Simulation of the spectral density function for the N-15 resonance and its temperature dependence using the Lipari-Szabo forma lism suggests that the dominant time scales of loop motion range from 0.6 to 18 ns. For comparison, the time scale for molecular rotation of the 70 kDa Hsp10 heptamer is estimated to be 37 ns. Complex behavior of the T-2 relaxation time indicates that motion also occurs on longer time scales. All of the modes of loop motion are likely to have an im pact on Hsp10/Hsp60 interaction and therefore affect Hsp10/Hsp60 funct ion as a chaperonin.