Structural Basis of Interactions Within and 
Between Macromolecules 
Brian W. Matthews, Ph.D., D.Sc. — Investigator 
Dr. Matthews is also Professor of Physics and Director of the Institute of Molecular Biology at the Univer- 
sity of Oregon and Adjunct Professor of Biochemistry at the Oregon Health Sciences University, Portland. 
He received his undergraduate and graduate training at the University of Adelaide, Australia. He did 
postdoctoral research at the MRC laboratory of Molecular Biology, Cambridge, England ( with David 
Blow) and at the National Institutes of Health ( with David Davies). Dr. Matthews is a member of the 
National Academy of Sciences. 
OUR laboratory' uses x-ray crystallography, in 
concert with other techniques, to address 
some of the fundamental problems in biology: 
How do proteins spontaneously fold into their 
biologically active three-dimensional configura- 
tions? What determines the stability of these 
folded proteins, and can stability be improved? 
How do proteins interact with each other? How 
do proteins interact with DNA? How do enzymes 
act as catalysts? 
The Protein Folding Problem 
An area of long-standing interest is the so-called 
protein folding problem. How does a newly syn- 
thesized, extended peptide chain "know" how to 
fold spontaneously into its active three-dimen- 
sional shape? 
Although it has long been recognized that the 
amino acid sequence of a protein determines its 
three-dimensional structure, recent work from 
several laboratories has made it clear that certain 
amino acids are more important than others in the 
folding process. At some positions, typically the 
solvent-exposed mobile sites in the folded pro- 
tein, amino acids can be interchanged at random 
with little apparent effect on folding or stability. 
These amino acids seem to be unimportant in 
protein folding. On the other hand, interchange 
of amino acids in buried or rigid parts of a folded 
protein can destabilize it, suggesting that the 
amino acids at these positions are important in 
determining the folded conformation. 
To try to simplify the complexity of the folding 
problem, an attempt has been made to replace 
some of the "nonessential" amino acids with ala- 
nine in phage T4 lysozyme. Such a "polyalanine" 
protein would, in principle, truncate all nones- 
sential side chains and permit focus on those 
parts of the amino acid sequence that are critical 
for the folding process. 
As a first step, a series of alanines was intro- 
duced within the a-helix that includes residues 
1 26- 1 34 of T4 lysozyme. The somewhat surpris- 
ing result was that alanines were not only toler- 
ated at most positions in the a-helix; they actually 
increased the protein's stability. This indicates 
that alanine is a strongly helix-favoring residue. It 
also suggests that the replacement of solvent- 
exposed residues of a-helices with alanines 
might provide a means of increasing the stability 
of other proteins. Finally, the fact that a series of 
alanines can be introduced into T4 lysozyme con- 
firms that this might be a way to simplify the pro- 
tein folding problem. 
Engineering Proteins of Enhanced Stability 
We are using the lysozyme from bacteriophage 
T4 to define the contributions that different types 
of interaction (hydrogen bonds, hydrophobic in- 
teractions, salt bridges, etc.) make to the stability 
of proteins. Much of our emphasis during the past 
year has been on gaining a better understanding 
of electrostatic interactions. It is known that they 
can be strong in some cases but seemingly weak 
in others. To try to explain this, we first investi- 
gated the effect of long-range electrostatic inter- 
actions by using genetic engineering to change 
the charge of a number of groups on the surface 
of phage T4 lysozyme. We were able to reduce 
the overall charge on the protein from +9 units to 
+ 1 unit. Nevertheless, there was almost no 
change in protein stability, indicating that the in- 
teraction between the different charged groups is 
very weak. 
Next we examined short-range electrostatic in- 
teractions by introducing negatively charged 
groups adjacent to positively charged amino 
acids on the surface of the protein. Again, there 
was almost no change in the protein stability. 
Finally, we introduced charged groups next to 
the ends of a-helices. In this case an increase in 
stability was consistently observed, indicating 
that there are favorable electrostatic interactions 
between charged amino acids and the charges on 
the ends of the a-helices. This is the so-called 
"helix dipole" effect. 
It is at first surprising that electrostatic interac- 
tions between charged groups on the surface of a 
protein tend to be weak, whereas interactions 
with the a-helix dipole tend to be much stronger. 
The reason for the difference, we believe, is that 
charged groups on the surface of a protein tend to 
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