Research Experience
Involvement with the UH Structural Biology group
I began working with
Dr. Kurt L. Krause soon after his arrival at the University of
Houston. My thesis project involves the crystal structure
determination of a novel nuclease from Serratia marcescens. This
project is particularly intriguing, since this nuclease is different,
both in primary sequence and in specificity, from the other nucleases
whose structures are known. I have been closely involved in many
aspects of the project to date, including protein purification, crystal
growth, data collection, heavy atom screening, Patterson solution, heavy
atom refinement, and model building. The first task was the
purification of the protein from the genetically modified strain of
S. marcescens. We had some initial trouble with
microheterogeniety resulting from two isoforms of the nuclease being
present. Once these isoforms were separated, it was possible to
determine the optimal conditions to grow large single crystals. We have
reported large, high quality crystals using ammonium sulfate, (Miller
et al., 1991. J. Mol. Bio. 222: 27-30). The
nuclease crystals are orthorhombic, with cell dimensions a=106.7 Å
b=74.5 Å c=68.9 Å and they diffract to beyond 2 Å.
Heavy atoms derivatives have been used in multiple isomorphous
replacement (MIR) refinement, including platinum, lead, iridium,
mercury, and uranium. The crystals exhibit pseudo-I222 symmetry, as
indicated by Patterson and heavy atom refinement analyses. The crystals
are in the P2(1)2(1)2 space group with two monomers in the asymmetric
unit, but there is some ambiguity as to whether true twofold is down the
b or c axis due to the pseudo-centering. Similar quality electron
density maps can be calculated using best phases refined in either space
group for one of the monomers, but a diminution in electron density for
the non-crystallographically related monomer after refinement in
P2(1)22(1) suggests the current space group [P2(1)2(1)2) most accurately
reflects the packing. It was not possible to build the best MIR map
with the ammonium sulfate crystals. New crystallization conditions were
then determined using PEG 4000 as the precipatant. New heavy atom soaks
were then undertaken, and this led to good quality maps.
My senior honors thesis covered the initial part of this project. My
doctoral dissertation covers the final structure determination.
Worms Worms Worms ...
Prior to working for Dr. Krause, I had been involved with a wide variety of
research projects. My first position as an undergraduate research assistant
was with Dr. Ralph Hecht at the University of Houston. Here I worked
extensively with the model developmental system of the nematode
Caenorhabditis elegans. One of my major accomplishments was
developing a protocol which allows the screening of very large numbers
of mutagenized temperature sensitive mutants in order to find rare
suppressors. I was also involved with genetic mapping using classical genetic
crosses and physical mapping experiments using the micro-injection of DNA
into oocyte nuclei. Another series of projects involved computational
molecular biology. I preformed much of the data analysis for the
sequencing of the glyceraldehyde-3-phosphate dehydrogenase gene from
Thurmus aquatics and the study of evolutionary divergence of
two different species of nematode. I also assisted with a number of
sequencing and molecular biology experiments.
Summer internship
I spent one summer working for Dr. P. Michael Connelly in the family
studies division of the Dept. of Medical Genetics
(Indiana University Medical Center). Here I worked on correcting
database problems and with the compilation of statistical reports on
several genetic disease databases, including the Huntingtons Disease
database.
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Last Change: 29 August 1994 Mitch@bragg1.bchs.uh.edu