eral laboratories has revealed 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 protein, 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, sug- 
gesting that the amino acids at these positions are 
important in determining the folded conformation. 
To try to simplify the complexity of the protein- 
folding problem, Xue-jun Zhang and Dr. Dirk Heinz 
have attempted to replace "nonessential" amino 
acids in bacteriophage T4 lysozyme with alanine. 
Such a "polyalanine protein" would, in principle, 
truncate all nonessential side chains and bring into 
focus those parts of the amino acid sequence that are 
critical to the folding process. 
In experiments to date, a series of alanines has 
been introduced within two different a helices of 
T4 lysozyme. The somewhat surprising result is that 
alanines are not only tolerated at most positions in 
the a helix, they can sometimes increase the pro- 
tein's stability. In an extreme case it has been found 
that 10 alanines can be introduced in sequence, yet 
the protein still folds normally and has full activity. 
This illustrates that the information 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 stabi- 
lizing the folded structures of globular proteins is 
the hydrophobic effect. A principal difficulty in 
quantitating this effect has been the lack of relevant 
structural data. How does a protein structure re- 
spond when a bulky hydrophobic residue such as 
leucine is replaced by a smaller residue such as ala- 
nine? Does the protein structure remain essentially 
unchanged or is it rearranged to avoid the creation of a 
cavity? If cavities are created, do they contain solvent? 
To address these questions Dr. Elisabeth Eriksson 
constructed cavity-creating mutants in which a large 
hydrophobic amino acid was replaced by a smaller 
one within the hydrophobic core of bacteriophage 
T4 lysozyme. Several such variants were crystallized 
and the structures determined at high resolution. 
The structural consequences of the mutations dif- 
fered from site to site. In some cases the protein 
structure hardly changed at all. In other cases, both 
side-chain and backbone shifts up to 0.8- 1 .0 A were 
observed. In every case removal of the wild-type 
side chain allowed some of the surrounding atoms 
to move toward the vacated space, but a cavity al- 
ways remained. 
This suggests a way to reconcile the different val- 
ues for the apparent strength of the hydrophobic 
effect. Two extreme situations can be imagined. In 
the first, a leucine alanine replacement is con- 
structed, and the protein structure remains com- 
pletely unchanged. Here the size of the created cav- 
ity is large, and the mutant protein is maximally 
destabilized. In the other extreme, the protein 
structure relaxes in response to the leucine ala- 
nine substitution, fills the space occupied by the 
leucine side chain, and so avoids the formation of 
any cavity whatsoever. In this case the decrease in 
energy of the mutant protein relative to the wild 
type drops to a constant energy term that is charac- 
teristic for a leucine ->■ alanine replacement. 
Ligand Binding Within Cavities 
Dr. Eriksson and Dr. Walt Baase also showed by 
crystal lographic and thermodynamic analysis that 
the cavity created by the replacement leucine 99 
alanine in T4 lysozyme is large enough to bind ben- 
zene and that ligand binding increases the melting 
temperature of the protein by 5.7°C. This shows 
that cavities can be engineered within proteins and 
suggests that such cavities might be tailored to bind 
specific ligands. The binding of benzene at an inter- 
nal site 7 A from the molecular surface also il- 
lustrates the dynamic nature of proteins, even in 
crystals. 
Receptor-Ligand Interaction 
To develop an understanding of the mode of ac- 
tion of growth factors and their interactions with 
their receptors. Dr. Eriksson and Dr. Larry Weaver, 
in collaboration with Dr. Lawrence Cousens (Chiron 
Corp.), crystallized, determined, and refined the 
high-resolution structure of human fibroblast 
growth factor. The structure is very similar to that of 
interleukin-ljS. Clearly, many growth factors have 
similar overall structures, but the exact relationship 
of these factors in the vicinity of their receptor- 
binding regions remains to be clarified. 
Protein-DNA Interaction 
Dr. Matthews and his colleagues have been inter- 
ested for some time in the interaction between pro- 
teins and nucleic acids. In 1981 they determined 
the structure of the Cro repressor protein of X bacte- 
riophage (bacteria-infecting virus) . Cro has served 
as one of the prototypical examples of a DNA- 
interacting protein. Drs. Richard Brennan and Ste- 
ven Roderick, in collaboration with Dr. Yoshinori 
Takeda, have also determined the crystal structure 
of Cro in complex with a tight-binding 1 7-base pair 
DNA operator and are improving the accuracy of 
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