Student Theses and Dissertations

Date of Award

1996

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Chait Laboratory

Abstract

Powerful new techniques in mass spectrometry (MS) provide an unprecedented opportunity to develop methods that facilitate protein characterization. This thesis utilizes matrix-assisted laser desorption/ionization and electrospray Ionization MS to explore protein structure. Starting at the 'primary' level of protein structure, an approach is developed to aid in the characterization of posttranslational modifications and processing of mature gene products. The approach involves the elution of picomole amounts of whole proteins from SDS-PAGE gels in a form suitable for MS analysis. Application of the elution method to the obesity factor leptin led to the characterization of the endogenous form of leptin in obese humans. A biochemical procedure is developed that permits 'higher' order structural features of proteins to be probed. The method uses limited proteolysis to distinguish regions within a target protein that are solvent exposed and flexible from those that are solvent inaccessible or conformationally rigid. The DNA binding protein Max was investigated by the procedure and several structural features were determined. The structural study of Max was extended in an investigation of the effects of N-terminal phosphorylation. Limited proteolysis was found to be of great utility in the determination of the 'precise' boundaries of protein folding domains. This method of 'domain elucidation' is explored fully and applied to proteins of unknown structure, including the Drosophila TAF 42/62 protein complex. Limited proteolysis/MS experiments permitted a rapid design of a compact construct of the TAF complex, crystals of which X-ray diffracted to 1.4 Å resolution. MALDI-mass spectrometric methods were absolutely crucial to every step of the described research. However, before the methods could be effectively applied, it proved necessary to develop new and practical MS sampling handling procedures.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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