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The tryptophan repressor protein regulates intracellular concentration of Tryptophan in Escherichia coli by binding to DNA operators and is activated in the presence of high L-Trp concentration by formation of an L-Trp-bound holo-repressor. A Leu to Phe mutation at position 75 generates a temperature-sensitive mutant of TrpR, L75F-TrpR, whereas an Ala to Val mutation only two residue positions further on the protein sequence, at residue position 77, generates a super-repressor mutant of TrpR. Backbone amide dynamics studies on TrpR and the two variants using ¹â��µ N-NMR relaxation techniques at a magnetic field strength of 600 MHz (¹ H Larmor frequency) indicate that all three repressors exhibit comparable diffusion properties, implying that they exhibit very similar global shape, structure, and rotational diffusion properties in both apo- and holo- states, in solution. However, internal backbone amide dynamics of the three apo-repressors reveal small but significant differences in flexibility, which are found primarily for residues spanning the Helix-Turn-Helix DNA-binding domain. These results indicate that the fine-tuning of L-Trp binding interaction is modulated in different ways via small but significant changes in protein flexibility in the two TrpR variants in apo and L-Trp bound forms. Sulfolobus solfataricus, a model organism for Archaea, lives in extreme thermal and acidic environments such as the hot springs of Yellowstone National Park, and is host to diverse archaeal viruses including Sulfolobus spindle shaped virus-1 (SSV1) and Sulfolobus spindle shaped virus-Ragged Hills (SSV-RH). SSV viruses exhibit remarkable morphology and genetic diversity, but are poorly understood as many proteins encoded by their genomes have very little sequence homology to proteins of known functions. We have performed detailed backbone dynamics studies to better understand the mode of ligand recognition by E73, a 73-residue, homodimeric protein encoded within SSV-RH genome. Analysis of backbone dynamics measurements obtained for E73 provides evidence for fast time scale dynamics in the proposed nucleic-acid binding site and motion on the microsecond to millisecond time scale in the loop connecting helices alpha A and alpha B. |
