It is proposed to examine the folding process of as many enzymes as can be obtained in reasonable quantity and purity from
thermophilic bacteria, with an emphasis on the use of affinity chromatography. This technique which my colleagues, Cuatrecasas
and Wilchek and I developed in 1968 while at the National Institutes of Health should be of great value in these experiments
since it can be employed not only for studying the formation and binding properties of active centers, but also as a convenient
method for purification. We have, in the past, been able to purify a number of proteins from crude extracts to homogeneity
in one step because of the highly specific nature of active site binding to a unique ligand designed to catch the enzyme in
question. Although a considerable number of proteins from thermophiles have been isolated and studied in terms of their activity
and stability in the high temperature range, there has been very little on the chemical nature of the enzymatic binding sites
in terms of the portions of the peptide chains involved and the nature of amino acid replacements in going from mesophilic
to thermophilic enzyme species. If the proposed experiments work out well, we should be able to add some interesting and
useful information to the nature of the side chain interactions that lead to tertiary structure stabilization at elevated
temperatures. It has been pointed out that a significant understanding of protein folding and active center formation in
thermophilic enzymes will probably eventually require crystallographic analysis of the three-dimensional structure. It has
been shown by Thomas Powers and his colleagues, Frederic Richards and Harold Wykoff, that X-ray crystallography can be carried
out using the more or less standard procedures on crystals at high temperatures. It has also been found by Zierer, et al.,
at the Max Planck Institute for Molecular Genetics that thermophilic proteins can be crystallized in the usual manner. A
DNA binding protein from Thermus thermophilus was isolated from cells grown at 75 degrees C and, following purification, formed
good hexagonal crystals which diffracted to at least 3 angstroms. Comparison of such a structure in the presence and absence
of DNA should be of considerable interest.
Affinity columns are being prepared employing the procedures of modification of silica by glycidoxylation followed by periodate
oxidation and attachment of the ligand of interest, generally separated from the backbone by an aminohexyl "arm."
Such columns have worked quite well in HPLC equipment although the temperatures so far examined have not exceeded the usual
room temperature conditions. Testing and further development of such columns will undoubtedly require a bit of experimentation.
It is hoped to have columns prepared that are suitable for several dehydrogenases and for beta galactosidase within the next
few months, and the preparation of such columns will be facilitated by the kind assistance of experts at the Milligen Corporation
who have offered to make available their column packing equipment.
Since crystallography does appear to be a very desirable adjunct to the studies of structure and function, we will attempt
to select a particularly favorable enzyme in terms of its quantity in the bacterial cells and its ease of purification to
prepare quantities sufficient for crystal preparation and diffraction pattern examination. A colleague in this department,
Professor Evangelos Moudrianakis, as well as an active crystallographic group in the Biophysics Department nearby will assist
us with advice and equipment when this stage is reached.
There is a possibility that the work that is carried out might be of some value in industrial application. The use of thermostable
enzymes in the processes of oxidation, reduction, hydrolysis or introduction of substituents on aromatic and other backbone
structures will be examined.
The overall program will clearly involve the standard techniques of protein chemistry including amino acid analysis, fluorescence
and spectrum determination.