Principal Investigator Frank Gibson Project g11

Membrane Biochemistry Group, Machine VP

John Curtin School of Medical Research


A Combined Molecular Biological and Computer Graphics Study of Biologically Active Proteins

Traditional biochemical techniques have proved inadequate to study the structure and function of the physiologically important group of membrane-bound proteins. However the advances in molecular biology have allowed the amino acid sequences of a great number of such proteins to be determined and the introduction of "site-directed mutagenesis" allowing the replacement of specific amino acid residues has facilitated the study of protein function by comparing normal and "mutant" proteins.

The use of computer graphics is playing an increasingly important role in such studies, allowing visualization of possible structures. Of the 3000 or so amino acid sequences classified under the heading "membrane" in the Swiss-Protein Data Bank, only about half a dozen high resolution structures have been determined. This is due principally to the difficulty of isolating and crystallizing such proteins.

Most of our molecular graphics work is carried out in collaboration with laboratory workers who are studying proteins for which no crystallographic or nuclear magnetic resonance data is available. Possible structures can often be predicted, by appropriate algorithms, for portions of the proteins. In particular the membrane imbedded sections which are likely to have a helical structure. The modelling of these structures facilitate the design of experiments, using site-directed mutagenesis, to further the study of protein function.

A number of proteins of diverse source and function have been under investigation during the past year. These include the proton-translocating ATP synthase, an interest in our laboratory for many years, and variety of other proteins concerned with the transport of water soluble compounds across the lipid layers of the cell membrane. Molecular graphic representations of the "GABA receptor", a chloride-selective channel important in the transmission of nerve impulses, and some viral proteins, have proved particularly useful in postulating structure-function relationships.


What are the basic questions addressed?

How do the possible structures for the proteins under investigation relate to their known functions?

What are the results to date and future of the work?

Work on the structures of the Fo subunits of the ATP synthase has continued and the model elaborated. The details are a matter of debate and further modelling will be carried out. Two of the transmembrane helices of the GABAA subunit of the GABA receptor have been modelled and the results combined with those of laboratory studies have allowed the proposal of functional roles for various side chains of the amino acids of the helices. The Vpr protein of the human immunodeficiency (HIV) virus has been shown to form a cation-selective channel channel. This small protein has a series of negatively charged amino acids at the N-terminal end and positively charged amino acids at the C-terminal end. The initial structure formed by modelling the protein with an a-helix predicted to be in the protein is shown in Fig 1a. Upon extensive minimization the structure folds into a structure (Fig. 1b) which is stabilized by salt bridges. To obtain evidence as to whether the modelled structure corresponds to the structure in the cell it is possible to test the effect of replacing the charged amino acids on the behaviour of the protein in vivo.










Fig.1 a shows the backbone of the Vpr protein as originally modelled with relevant positively (+) and negatively (-) charged amino acid residues represented as light and dark space-filling structures respectivel.y.

Fig.1b shows the same sequence after extensive minimization.

Modelled structures have been presented in two PhD theses and a paper (see publications).

What computational techniques are used and why is a supercomputer required?

The commercial molecular modelling packages Insight and Homology from Biosym is being used for modelling and their program Discover for energy calculations. The simulation of the structures in which we are interested require extensive calculations for energy minimizations and molecular dynamics. A typical energy minimization requiring 2 h on the VP2200 requires about 100 h on our Iris workstation. The use of the supercomputer not only allows many more structures to be examined but frees the Iris for further modelling and computations on smaller molecules

Publications

Structure-Function Relationship of the Human GABAA Receptor, M. L. Tierney, Ph.D. Thesis, Australian National University (1995)

Molecular Studies on the Energy-Transducing ATP Synthase, A. J. Rodgers, Ph.D. Thesis, Australian National University (1995)

Vpr Protein of human immunodeficiency virus type 1 forms cation-selective channnels in planar lipid bilayers, S.C., Pillar, G.D., Ewart, A.Premkumar, G.B.Cox and P.W. Gage, Proc. Nat. Acad. Sc. USA (in press)