Model Studies of Protein Folding and Stability.

Our model studies of protein folding and stability have focused on understanding the role of backbone-backbone H-bonds in the folding and stability of two model multimeric proteins including the homo-hexameric 4-oxalocrotonate tautomerase enzyme and the homo-dimeric P22 bacteriophage Arc repressor protein. Backbone-backbone hydrogen bonds involving C=O and NH groups constitute a large number of the native contacts in folded proteins.  However, relatively little is known about the magnitude of their contribution to protein folding and stability.  This is largely because backbone mutations cannot be systematically introduced into proteins using conventional site-directed mutagenesis protocols that rely on recombinant DNA techniques.  The chemical synthesis strategies employed in our work enable the study of enzyme analogues with modified peptide backbones.

SUPREX: A New Technique for Assaying Protein Stability.

A major part of our research effort has focused on the development and application of a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for measuring the thermodynamic stability of a protein. The technique, which we have termed SUPREX (Stability of Unpurified Protein by Rates of H/D Exchange) removes many of the limitations currently associated with protein stability measurements. In contrast to conventional methods, the SUPREX technique is amenable to automation and high throughput analyses; and it is useful for the analysis of mmol to pmol quantities of both pure and impure protein samples.  We are currently using the SUPREX technique to study a variety of different protein and protein-ligand systems.

The core analytical protein methods that we employ in our fundamental and practical studies of protein folding and stability include: matrix assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry, reversed-phase HPLC, size exclusion chromatography, circular dichroism spectroscopy, and protein H/D exchange techniques.  In our studies we also employ solid-phase peptide synthesis strategies for the total chemical synthesis of proteins and for the construction of combinatorial peptide libraries that contain both natural and unnatural amino acids.

Major equipment in the Fitzgerald Laboratory includes: a Voyager DE MALDI-TOF Biospectrometry Workstation from PerSeptive Biosystems, Inc.; a API 150Ex single quadrupole ESI mass spectrometer,  two HPLC systems (including a Rainin dual-pump high pressure mixing system with UV detection and a Rainin Gradient Protein Chromatography System with PEEK parts.  The laboratory is also equipped with the infrastructrure to support solid phase peptide synthesis using Boc-chemistry. 

contact: michael.c.fitzgerald@duke.edu
last updated August 24, 2002