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John Bradley

  • B.Sc, Ph.D.
  • Temporary Analyst at the Medical Bureau of Road Safety, Dublin, Ireland
  • University College Dublin
  • 9Followers
  • 8Following

Research interests

Protein Stability

About

I'm interested in most aspects of Biology, that is, Molecular Biology, Biochemistry, Genetics, and the applications of Bioinformatics. But I also maintain an interest in many other areas of science such as Neuroscience, and Evolution. My research has changed over the years as I've progressed through my PhD, I've worked on, 1. Investigating Amyloid Fibre Formation. The initial topic of my PhD was to investigate the kinetics of amyloid fibre formation in hen lysozyme. Low pH and high temperatures were used to push the natively folded protein to the irreversible and highly stable misfolded state. Kinetics were followed using ThT fluorescence and SDS-PAGE was used to follow loss of native protein. 2. Assessing The Role Of Aspartic Acids In The Stability & Enzymatic Activity. This work stemed from the earlier Amyloid investigations. It became obvious that Aspartic Acids were an important part of the proposed mechanims in fibre formation. There are seven Aspartic Acids in HEWL, each of these were mutated to Alanine, along with some double and tripple mutants. It was expected that removal of these Aspartic Acid residues would result in a fragmentation resistant protein at low pH. Effects of the mutations on the stability of the protein were measured using CD-Sepctropolarimetry, SDS-PAGE, Enzymatic Assays, and Mass Spectrometry, MALDI-TOF, for a selection of single, double and tripple mutants. However, more work is required to draw definitive conclusions. To this end I hope to finish this work using 6-fold or ->7-fold Asp->Ala mutants. 3.Chemical Denaturation Of Hen Lysozyme Chemical denaturation of proteins provides a very controlled and accurate means of determining themodynamic paramneters of entopy, enthalpy, and ultimately, deltaG values. I investigated the denaturation behaviour of WT-HEWL using Guanidine Hydrochloride. The aim was to obtain good quality data for thermodynamic analysis, however, this proved to be experimentally in-practical, and for this reason, I switched to thermal denaturation studies of lysozyme. 4.Current Work - Thermal Denaturation Of HEWL and Mutant Variants My current work is focused on thermal denturation of HEWL with the aim of generating pH stability profiles for the WT and selected mutants. In particular, the effects of removing six individual Aspartic acids have been studied, along with a series of double and triple mutants. Values of entropy, enthalpy and delta-G for each mutation were obtained over a wide pH range for each mutant protein, and measured against predicted values. As there is a correlation between delta-G and delta pKa, this data set can also be used to generate an approximation of the pKa value for each Aspartic acid residue.

Groups

Professional experience

Temporary Analyst at the Medical Bureau of Road Safety, Dublin, Ireland

University College Dublin

November 2012 - Present

Ph.D Student

University College Dublin

October 2006 - August 2011(5 years)

Clinical Data Coordinator

Quintiles

February 2006 - September 2006(7 months)

Education

Doctorate in Protein Biochemistry

University College Dublin

October 2006 - August 2011(5 years)

Biochemistry

Department of Biochemistry, University College Dublin

September 2001 - June 2005(4 years)