J. Eric Gouaux

Current Institution
Oregon Health & Science University
Senior Scientist
Vollum Institute

Scholar: 1994

Awarded Institution
The University of Chicago


Research Interests

Elucidating molecular mechanisms to describe biological activity

The research in my laboratory is focused on elucidating molecular mechanisms to describe biological activity and physical properties of integral membrane receptors, enzymes and channels. Three-dimensional structures are determined by x-ray crystallographic techniques, modified forms of the molecules are generated by site-directed mutagenesis and selected biophysical and biochemical techniques are employed to measure physical and functional properties. Our studies are roughly divided into three areas. The central area concerns the crystallographics study of membrane proteins, alpha-hemolysin, (alphaHL) representing the profect which is the farthest along; a second area is devoted to the development of methods for membrane protein crystallization; and a third area involves biophysical studies on bacteriorhodopsin.

X-ray crystallographic studies of integral membrane proteins. The pathogenic action of infectious agents, the transduction of energy, the active and passive transport of ions and molecules, and the transmission of biochemical signals into and out of cells and organelles are some of the biological processes mediated by membrane proteins. Models for the folding, function and stability of membrane proteins are much less developed when compared to models that describe the corresponding properties of water-soluble proteins. Although substantial progress has been made in the cloning, expression and characterization of important membrane proteins, a complete understanding is precluded by a lack of atomic resolution structures. This is in part due to the difficulty of obtaining well-ordered, three-dimensional crystals as well as being due to limitations in over-expression and purification.

AlphaHL as a model for oligomeric transmembrane channels. AlphaHL provides an excellent model system for elucidating the molecular mechanisms of protein insertion into a membrane, of folding and assembly of a defined multimeric aggregate, and of formation and regulation of a transmembrane pore by divalent cations. AlphaHL, one of the important virulence factors from Staphylococcus aureus, is secreted as a water-soluble monomer of 293 amino acids that is synthesized with a 26 amino acid signal sequence. Upon encountering a susceptible membrane, such as that provided by a rabbit erythrocyte, the monomer binds to membrane, undergoes membrane insertion, and oligomerizes to form a heptameric transmembrane pore.

Over the last two years we have worked on crystallization and structure determination of monomeric and heptameric forms of alphaHL. We have obtained many distinct crystal forms of the detergent-solubilized heptamer and the water-soluble monomer. Application of our crystallization strategies and the use of amphiphiles have resulted in the growth of crystals of the heptamer that diffract to 1.8 Angstroms using a laboratory x-ray source and to 1.6 Angstroms using a synchrotron x-ray source. At the present time, we are completing the refinement of the heptameric form, at 1.9 Angstroms resolution, and have begun work on a manuscript. The structure is one of a few membrane protein structures determined to high resolution and is the first structure of a toxin in its oligomeric, membrane active form.

Methods for membrane protein crystallization. Elucidation of high resolution structures of membrane proteins is limited by the availability of well ordered crystals. The major problem in membrane protein crystallization is to maintain the protein in a detergent-sobulized form that does not disrupt the native conformation of the protein and that will still allow for crystal formation. Frequently, the conditions which stabilize the native, biologically active form of the protein are not the same as those which are favorable for crystallization. Nonetheless, once conditions for stability are satisfied. then one must search for conditions which will promote crystal formation. The thorough exploration of this multidimensional space - defined by such variables as precipitant type and concentration, pH, temperature, and ion type and concentration, to mention a few - is crucial to the success of crystallization experiments.

Bacteriorhodopsin, a model for membrane protein folding and stability. There is very little information on the relationship between the amino acid sequence and the folding and stability of integral membrane proteins. We have chosen bacteriorhodopsin (bR) as a model system for investigating the effects of amino acid substitutions on the kinetic and thermodynamic stability of helical membrane proteins. In addition, we are carrying out experiments to determine the extent to which a membrane protein might be engineered in an effort to facilitate crystallization.

Toward these ends, we have designed and synthesized a gene for bR and have expressed the protein, at high levels, in E. coli. The protein has been purified to homogeneity and regenerated with its cofactor, all-trans retinal. We are currently in the process of studying the effects of temperature and various reagents, such as urea and sodium dodecyl sulfate for example, on the denaturation of bR in an effort to determine whether there are conditions under which bR will reversibly unfold and renature in a cooperative, two-state fashion.

In an effort to discover whether we could use molecular biology to facilitate the crystallization of membrane proteins, and to investigate the properties of membrane proteins in general, we have altered the hydrophobic, lipid-exposed surface of helix aspartic acid by replacing residues with polar or charged amino acids. On the basis of these experiments we have found that alteration of amino acids 113, 116, 117, 120 and 124 to glutamine, glutamine, aspartic acid, glutamine and glutamine respectively, gives a bR molecule which folds and regenerates with retinal to a species with nearly wild-type spectral properties.