Backbone Dynamics in an Intramolecular Prolylpeptide SH3 Complex from Diphtheria Toxin Repressor, DtxR

• Other Authors: Bhattacharya, Nilakshee (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biochemistry; Biophysics; Molecular biology
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-1408

Diphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined experimentally using NMR spectroscopy and computed from molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting b-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment also exhibited low order parameters with internal rotational correlation times on the order of 0.6 – 1 ns. Further analysis showed that the SH3 domain was rich in ms motions while the Pr segment was rich in motions on the 100s ms timescale. Molecular dynamics trajectories of PrSH3 and SH3 indicated structural rearrangements that might contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The intramolecular complex resisted any significant change in the binding affinity between the Pr segment and the SH3 domain due to mutations in the Pr segment. The results of this study provide key insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity. === A Dissertation Submitted to the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. === Fall Semester, 2007. === October 23, 2007. === NMR, SH3 domain, relaxation, Lipari-szabo === Includes bibliographical references. === Timothy M. Logan, Professor Directing Dissertation; Huan-Xian Zhou, Outside Committee Member; Hong Li, Committee Member; Oliver Steinbock, Committee Member.