TrHbs: Biophysical Consequences of a Nonpolar Tunnel

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DESCRIPTION (provided by applicant): Truncated hemoglobins (trHbs) are a recently discovered class of small oxygen-binding protohemeproteins, widely distributed in prokaryotes. Many occur in bacteria pathogenic to man (e.g. Mycobacterium tuberculosis (TB), Mycobacterium avium, Mycobacterium leprae, Cornybacterium diphtheriae, Bordetella pertussis, Legionella pneumophila, Staphylococcus aureus and Bacillus anthracis). This project will focus on trHbN and trHbO, the two trHbs from TB. TrHbN protects aerobic respiration from the inhibitory action of nitric oxide and enhances infectivity. The function oftrHbO is essential for bacterial growth. Since trHbs are not present in man, this makes them potential drug targets. The emphasis of this project is on probing those unusual biophysical properties of these two trHbs that are hypothesized to be the basis for the unusual functionalities that are of potential biomedical significance. Several specific aims are designed to expose the nature and significance of the hydrogen bonding network found in the distal hempocket and of the large apolar tunnel that links the solvent to the ligand binding site at the heme. To achieve in depth insight into how the hydrogen bonding network and the apolar tunnel functioning of these two important proteins, several techniques will be used including photolysis, stopped-flow spectrometry, vibrational spectroscopy (FTIR, UV and visible resonance Raman), structure analysis (X-ray diffraction, NMR), site directed mutagenesis and conformational trapping and kinetics of ligand rebinding. In parallel with this part of the overall project, genetic and molecular biological approaches will be used to correlate the biophysical results with the in vivo functional studies.