Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (ii) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca 2؉ is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca 2؉ , possibly a (MboII) 2 -(DNA) 2 complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme) 2 -(DNA) 2 complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.The best characterized type II restriction endonucleases, exemplified by EcoRI, EcoRV, BamHI, and PvuII, are homodimers that cut DNA within palindromic target sites, from 4 to 8 base pairs in size (1-3). Generally one protein subunit recognizes one-half of the palindromic sequence and, in an identical arrangement, the second subunit interacts with the other half, resulting in a symmetric protein-DNA complex. Each of the protein subunits contains a catalytic site, enabling both strands of the DNA to be cut, often in a highly concerted reaction. However, many type II restriction endonucleases do not correspond to these simple paradigms. One category, classified as type IIs systems (4), recognizes nonpalindromic DNA sequences, between 4 and 7 base pairs in length and cut up to 20 bases outside their target sites. A nonsymmetrical DNA target site does not allow for simple recognition using a protein with a homodimeric subunit arrangement, and indeed, most type IIs restriction enzymes appear to be monomeric (5-7). As a monomeric enzyme will contain only a single active site, there is no straightforward way to achieve the hydrolysis of both DNA strands. FokI, the best studied type IIs enzyme, recognizes GGATG [9/13] sites (the numbers in brackets refer to the distance to the cutting site on the top and bottom strands, respectively) and contains separate DNA recognition and cutting domains (8). A crystal structure shows FokI bound to DNA as a monomer with the recognition domain interacting with the GGATG bases; the cutting domain is tightly associated with the recognition domain and too far from the scissile phosphates to cause hydrolysis (9). A second structure, of the free enzyme, shows a dimer in which the dimerization interface is com...

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