John Perona perona
Fri Jan 19 18:43:59 EST 1996


        An NIH-funded postdoctoral position is available to use a combination 
of enzyme kinetic and crystallographic approaches to explore specific DNA 
cleavage by the EcoRV endonuclease.  Our approach is to correlate pre-steady 
state kinetic measurements with crystal structures, for a variety of mutant 
enzymes exhibiting altered DNA binding and catalytic functions.  A parallel 
project is also underway which explores the use of genetic selection methods 
in E. coli for the isolation of EcoRV mutants with altered substrate 
specificity profiles.

        This project is intensively structure-based and provides a bridge for 
the classical enzymologist or protein crystallographer to acquire new training
at an interface where both perspectives are required.  Very few enzymes have 
been simultaneously studied by the combination of pre-steady state kinetics and 
crystallographic analyses of mutants.  We believe that this integrated approach 
has some substantial potential to provide a database of information crucial to 
rational re-engineering of existing protein scaffolds to new function.

        Some of the specific questions we will be exploring in the immediate 
future are: (1) the structural and energetic basis for indirect readout of DNA 
sequence information, particularly at the central TA step of the GATATC target
site where no discriminatory enzyme-DNA contacts are made; (2) the molecular 
determinants of the protein conformational change which occurs upon specific 
but not non-specific DNA binding; (3) the role of distal enzyme-DNA contacts in 
facilitating the individual binding and catalytic steps; (4) the extent to
which water-mediated enzyme-DNA interactions provide binding strength and 
specificity equivalent to direct contacts; (5) the structural pathway of
catalysis including the roles of several implicated divalent metal ions.

        A background in either enzymology or macromolecular crystallography is 
required for this position. Interested individuals should forward a CV to:

Dr. John Perona
Department of Chemistry
University of California at Santa Barbara
Santa Barbara CA 93106-9510
Tel. 805-893-7389       email: jperona at

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