Structural Basis of Interactions Within 
and Between Macromolecules 
f 
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 
University 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 try to ad- 
dress some of the fundamental problems in biol- 
ogy: How do proteins spontaneously fold into 
their biologically active three-dimensional con- 
figurations? What determines the stability of 
these folded proteins, and can stability be im- 
proved? How do proteins interact with each 
other? How do they 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- 
dimensional 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 almost at 
random with little apparent effect on folding or 
stability. 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. 
One of the encouraging developments has 
been the relative freedom with which amino acid 
replacements can be introduced in a protein of 
interest. To try to simplify the complexity of the 
protein-folding problem, we are attempting to 
replace some of the "nonessential" amino acids 
in phage T4 lysozyme with alanine. Such a 
"polyalanine protein" would, in principle, trun- 
cate all nonessential side chains and allow one to 
focus on those parts of the amino acid sequence 
that are critical for the folding process. 
In experiments to date, a series of alanines has 
been introduced within two different of-helices of 
T4 lysozyme. The somewhat surprising result is 
that alanines are not only tolerated at most posi- 
tions in the a-helix; they can sometimes increase 
the protein's stability. In an extreme case it has 
been found that 1 0 alanines can be introduced in 
sequence, yet the protein still folds normally and 
has full activity. This illustrates that the informa- 
tion in the amino acid sequence of a protein is 
highly redundant. 
Understanding the Interactions That 
Stabilize Protein Structures 
It is generally agreed that the major factor in 
stabilizing the folded structures of globular pro- 
teins is the hydrophobic effect. Until recently it 
has also been generally agreed that the strength of 
the hydrophobic effect — i.e., the energy of stabi- 
lization provided by the transfer of hydrocarbon 
surfaces from solvent to the interior of a protein 
— is about 25-30 cal mol~' for each square ang- 
strom of surface area buried within the protein. 
However, some recent studies using site-directed 
mutagenesis and protein denaturation have sug- 
gested that the strength of the hydrophobic effect 
might be much higher. 
A principal difficulty in addressing this prob- 
lem has been the lack of relevant structural data. 
How does a protein structure respond when a 
bulky hydrophobic residue such as leucine is re- 
placed by a smaller residue such as alanine? Does 
the protein structure remain essentially un- 
changed or is there structural rearrangement to 
avoid the creation of a cavity? If cavities are cre- 
ated, do they contain solvent? 
To address these questions, six "cavity-creat- 
ing" mutants in which a large hydrophobic 
amino acid was replaced by a smaller one were 
constructed within the hydrophobic core of 
phage T4 lysozyme. All variants were crystallized 
and the structures determined at high resolution. 
The structural consequences of the mutations 
differ from site to site. In some cases the protein 
structure hardly changes at all. In other cases, 
however, both side-chain and backbone shifts up 
to 0.8-1.0 A were observed. In every case re- 
moval of the wild-type side chain allowed some 
of the surrounding atoms to move toward the va- 
cated space, but a cavity always remained. 
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