Project Title

Specific Histidines Play A Role in pH Sensing

Presenters

Academic department under which the project should be listed

CSM - Molecular and Cellular Biology

Faculty Sponsor Name

Dr. Susan Smith

Abstract (300 words maximum)

James Scott, Emily Onyekwere, Susan M.E.Smith

The pH sensitive nature of dinoflagellate bioluminescence provides an excellent tool for studying the pH sensing mechanism of proteins. The dinoflagellate Lingulodinium polyedrum generates bioluminescence through the interactions of two proteins – luciferin binding protein (LBP) and the enzyme luciferase (LCF) – with the enzyme substrate luciferin (LH2). At pH8, LCF is inactive, and LH2 remains bound to LBP. When the pH drops to 6, LBP releases LH2, and LCF becomes active, catalyzing the oxidation of LH2 and producing light as a by-product. In LCF, specific histidines are known to confer the pH sensing that regulates activity. We hypothesize that one or more of the 9 histidines in LBP confer the pH sensing that controls LH2 binding. LBP in which pH sensing histidines have been mutated should be incapable of delivering LH2 to LCF, which can be detected in a luminescence assay. So far, site directed mutagenesis of the LBP coding sequence has resulted in alanines substituted at H167, H209, H211, H340 H347, H638, H639, and H483[SMS1] , with mutant H347A still to be made. Purified recombinant mutant and wildtype LBP preparations were “charged” by incubation with LH2 at pH 8, followed by sized exclusion chromatography to remove free LH2. Each mutant is tested in a luminescence assay to measure its ability to deliver LH2. Some mutants show less luminescence than wildtype, whereas other mutants show the same emission of light as the wild type. Thus, some but not all histidines affect LBP’s ability to sense pH.

Project Type

Poster

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Specific Histidines Play A Role in pH Sensing

James Scott, Emily Onyekwere, Susan M.E.Smith

The pH sensitive nature of dinoflagellate bioluminescence provides an excellent tool for studying the pH sensing mechanism of proteins. The dinoflagellate Lingulodinium polyedrum generates bioluminescence through the interactions of two proteins – luciferin binding protein (LBP) and the enzyme luciferase (LCF) – with the enzyme substrate luciferin (LH2). At pH8, LCF is inactive, and LH2 remains bound to LBP. When the pH drops to 6, LBP releases LH2, and LCF becomes active, catalyzing the oxidation of LH2 and producing light as a by-product. In LCF, specific histidines are known to confer the pH sensing that regulates activity. We hypothesize that one or more of the 9 histidines in LBP confer the pH sensing that controls LH2 binding. LBP in which pH sensing histidines have been mutated should be incapable of delivering LH2 to LCF, which can be detected in a luminescence assay. So far, site directed mutagenesis of the LBP coding sequence has resulted in alanines substituted at H167, H209, H211, H340 H347, H638, H639, and H483[SMS1] , with mutant H347A still to be made. Purified recombinant mutant and wildtype LBP preparations were “charged” by incubation with LH2 at pH 8, followed by sized exclusion chromatography to remove free LH2. Each mutant is tested in a luminescence assay to measure its ability to deliver LH2. Some mutants show less luminescence than wildtype, whereas other mutants show the same emission of light as the wild type. Thus, some but not all histidines affect LBP’s ability to sense pH.