Multiplex labeling: A new method for determination of membrane protein topology
Abstract
The multipolar nature of membrane bilayers makes structure determination of membrane proteins very difficult. In fact, except for relatively low resolution 2D diffraction results, no crystal structures of proteins in membranes are known. Structures have either been determined by crystallizing in detergents or they have been deduced by combining data from a variety of biophysical experiments. Thus a general method for structure determination of membrane proteins is needed. Cysteine mutagenesis has been used for many years to provide structural information, however, current applications are handicapped by the need to purify each mutant, complex sample preparation and the inability to interpret the results clearly. This thesis presents a new method of topological analysis via cysteine mutagenesis using four different fluorescent labeling methods on the same batch of over-expressing cells to provide topology, expression level and localization information without requiring purification of the mutants. The sample preparation requirements and interpretation of results are also simplified in the new method. Analysis is done via fluorescence scanning of SDS-PAGE gels. The method is used to propose a new topology for the Escherichia coli ribose transport protein RbsC via the preparation of 34 single cysteine mutants.
Degree
Ph.D.
Advisors
Hermodson, Purdue University.
Subject Area
Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.