Investigating Nucleotide Interactions of BdcA, a Rossmann-fold containing protein capable of biofilm dispersal 公开

Walsh, Shawn Isham (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/hh63sw059?locale=zh
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Abstract

Biofilms are a major problem in human health because they confer increased resistance to antimicrobial agents and adhere to medical devices. As a potential therapeutic, the protein BdcA was previously engineered to disperse biofilms, and proposed to function via the sequestering of cyclic dimeric GMP (c-di-GMP).1 This ubiquitous bacterial second messenger is known to control a variety of cellular processes including biofilm formation and dispersal. Unlike previously characterized c-di-GMP receptors, BdcA contains a Rossmann fold, which typically binds NAD(P)(H). We set out to characterize BdcA as the first of a potentially novel class of c-di-GMP receptors. However, we show that BdcA has no affinity for c-di-GMP. Rather, it binds nicotinamide adenine dinucleotide phosphate (NADPH). We have also created seven rational mutants of BdcA, and initiated isothermal titration calorimetry (ITC) and biofilm dispersal assays in order to correlate NADPH interactions with biofilm dispersal, thereby shedding light on the now enigmatic mechanism behind BdcA-mediated biofilm dispersal.

Table of Contents

Table of Contents

Introduction 1

Results and Discussion 4

1. Work based on c-di-GMP as the substrate for BdcA 4

2. Work based on NADPH-specific binding 11

Conclusion 18

Methods 19

1. Genomic Isolation and Cloning 19

2. Site-directed Mutagenesis 19

3. Protein Expression and Purification 20

4. Phosphodiesterase Activity Assay 20

5. HPLC Analysis of nucleotides 21

6. Sequence and Structure Alignments 22

7. Rapid Equilibrium Dialysis (RED) 22

8. Isothermal Titration Calorimetry 24

9. Biofilm Dispersal Assay 25

Works Cited 26

Supplemental Figures and Tables 28



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