Using stability mutagenesis and inverse methods for elucidating the structure of a bacteriophage chaperone

Adrienne Nikole Foster, Purdue University

Abstract

Protein structures have traditionally been determined by biophysical methods, including x-ray crystallography, nuclear magnetic resonance and electron micrograph image reconstruction. These methods directly determine protein structure; however, they can be expensive and difficult depending upon the properties of a given protein or protein complex. The time required and labor intensive nature of traditional techniques has exacerbated the disparity between the number of sequenced genes and the number of determined structures. Inverse methods for structural elucidation are expeditious in comparison to traditional techniques; however, they do not directly determine protein structure. Rather, inverse methods reveal features of a protein’s structure through agreement between predicted results with experimental observations. Progress in computational biology has advanced the ability to predict protein structure and often yields largely correct models among several alternative ones. Here, we discriminate between 3 predicted structures of bacteriophage λ tail fiber assembly protein (pTfa) using inverse methods based on the stability of site directed mutations. An amino terminal fragment of pTfa consisting of the first 108 amino acids (pTfa1-108) was solubly expressed in E. coli. Analysis by intrinsic fluorescence, analytical ultracentrifugation and circular dichroism suggest that pTfa1-108 is a cooperatively folded, monomeric, polypeptide composed of β-strands, β-turns, and unordered structure. In the absence of diffracting crystals of intact pTfa, three threading models of pTfa1-108 were predicted by the fold recognition programs FUGUE and 3D-PSSM. A series of mutations predicted to be differentially destabilizing under the different models were constructed. Changes in stability (ΔΔG D) due to mutation were determined by urea denaturation monitored by intrinsic fluorescence and compared to predicted ΔΔGD values for each model. A comparison of predicted ΔΔGD’s and experimental ΔΔGD’s reveals that the structure of pTfa may be similar to the substrate binding domain of the general ATP-dependent chaperone, DnaK, but also has characteristics reminiscent of the bacterial heme chaperone, CcmE. We also report two forms of intact pTfa. The two forms are characterized by their solubility in 1.0 M ammonium sulfate and remain differentially soluble for four months.

Degree

Ph.D.

Advisors

Friedman, Purdue University.

Subject Area

Molecular biology

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