Philip Bryan






Ph.D. Biochemistry, University of Tennessee and Oak Ridge National Laboratory, 1979


The efforts of Dr. Bryan's group in the past year have been to use genetic, biochemical and biophysical methods to the study subtilisin BPN' and streptococcal Protein G.

Subtilisin BPN' is a 275 amino acid serine protease secreted by Bacillus amyloliquefaciens. It is an enzyme of considerable industrial importance and has been the subject of numerous protein engineering studies by ourselves and others. Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. The in vivo production of native subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme.

The application of a battery of biophysical methods allow us to pursue two aims 1) Determination of the nature of the kinetic barrier in the uncatalyzed folding reaction. 2) Determination of how the propeptide acts to catalyze folding. Learning how to fold heterologously expressed proteins is one of the most vexing problems in biotechnology. The demonstration that the folding rate of subtilisin can be dramatically accelerated either by mutation or by intervention of the propeptide in folding process offers hope that other difficult protein folding problems might be similarly addressed, if the kinetic barriers to folding such proteins can be understood.


Streptococcal Protein G is a multi-domain component of the cell wall of some streptococcal species and binds to all subclasses of human IgG by the constant Fc region. The IgG binding function of Protein G is contained in a globular domain of 56 amino acids (GB). We have used this domain as a model 1) to study protein folding and 2) to study the basic mechanisms of molecular recognition involved in the high affinity interaction of GB with the heavy-chain, constant region (Fc) of IgG. The long term objective is to better understand the function of this medically important class of Fc receptor proteins and engineer new classes of Fc receptor. GB is an ideal protein to probe the relationship of amino acid sequence to structure and stability. It is on the lower limit in size for a stable unique protein structure. Furthermore it achieves its stability without disulfide bonds or tight ligand binding. Its small size permits the application of numerous biophysical techniques to study the folding and stability, in particular NMR methods. Little information is available concerning specific nature of the binding interaction of GB with Fc or the mechanism of its class selectivity. Because Protein G is class specific in its binding to IgG and binds to the portion of the immunoglobulin that defines its class, we believe that our studies will not only help elucidate the mechanism of recognition but also permit the engineering of Fc receptors with new class selectivities. Fc receptors with new properties will have numerous medical and biotechnological applications.