Molecular Recognition in Biological Systems and Bioinformatics

Structure of the P22 repressor amino terminal DNA binding domain in complex with DNA. The protein is represented as colored ribbons, DNA is depicted as skeletal backbone. Solvent molecules associated with the DNA are indicated as blue spheres.

DNA binding; kinetics; transcriptional regulation; molecular modeling; microbial pathogenesis.

The research in the Koudelka lab is focused around three central questions:

Mechanisms of Indirect Readout
The mechanisms whereby regulatory proteins recognize specific DNA sequences remains one of the most important areas of study in biology. This process requires that the protein be able to seek out and recognize its particular binding sequence, in the presence of an overwhelming number of potential non-specific binding sites. In our studies of direct readout of DNA sequence, we have uncovered the intimate details of how amino acids and base pairs can interact, and how these interactions can be regulated by both protein and DNA structure. In indirect readout, sequence-dependent differences in the structure and flexibility of noncontacted bases in a DNA binding site regulate the stability and sequence-specificity of a protein-DNA complex. Despite the high prevalence and functional importance of indirect readout it is unclear how DNA sequence differences lead to changes in DNA structure and flexibility. We are determining the structural basis for, and functional implications of the indirect readout mechanism used by bacteriophage repressor proteins.

Allosteric Effects of DNA Sequence on the Structure & Function of DNA Binding Proteins
DNA is more than a platform to which proteins bind. DNA sequence-dependent alterations of the tertiary and/or quaternary structure of many protein-DNA complexes modulate their gene regulatory activities. This is a second role for DNA in gene regulation, an allosteric effector of protein function. DNA-dependent differences in protein structure influence gene regulation by 1) changing the specificity of the protein-protein contacts within the protein-DNA complex; 2) altering the conformation of the complex formed between the DNA-bound protein and a protein recruited from solution and/or 3) modifying contacts between the proteins bound at adjacent sites.

Evolution of Bacteriophage-encoded exotoxins
Bacterially-derived exotoxins are among the most deadly substances known. The genes that encode these exotoxins are usually carried by bacterial viruses (bacteriophages) integrated into the bacterial host chromosome. It is generally assumed that the targets of these toxins are mammals. However, these phage-encoded exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions that lack the presumed mammalian targets. These observations suggest that humans and other susceptible mammals are not the primary "targets" of these toxins. We are exploring the hypothesis that exotoxins are part of an antipredator defense mechanism.

• AKTA Basic FPLC
• French press
• Kintek quenched flow apparatus

See LMVA (Laboratory for Molecular Visualization & Analysis)

• E. coli
• Lambdoid bacteriophages
• Tetrahymena

• Helix-turn-helix DNA binding transcription regulators
• Prokaryotic RNA polymerase
• RecA

• Microbial pathogenesis
• As related to bacterial dysentery
• Entherohemorrhagic Escherichia coli Infection

• Methods to assess/assay DNA binding
• Homology-based protein modeling
• Site-directed mutagenesis
• Protein overexpression and purification

Haemorrhagic colitis, infantile diarrhea, haemolytic uremic syndrome

http://jb.asm.org/cgi/content/full/187/16/5624?view=long&pmid=16077107

http://jb.asm.org/cgi/content/full/186/22/7659?view=long&pmid=15516580

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-4CG0J84-6-K&_cdi=6899&_user=681891&_orig=search&_coverDate=07/09/2004&_qd=1&_sk=996599996&view=c&wchp=dGLzVlz-zSkWA&_valck=1&md5=02bbfa02d377923ffa8c497c12030009&ie=/sdarticle.pdf

http://pubs.acs.org/cgi-bin/sample.cgi/bichaw/2003/42/i14/pdf/bi027318j.pdf