Ribonuclease Sa (RNase Sa) is an extracellular enzyme secreted by Streptomyces aureofaciens into the growth medium. It is the smallest member of the microbial T1 ribonuclease family with just 96 amino acids. Crystal structure of RNase Sa has been determined at 1.2 and 1.0 Å resolution [1,2] and a solution structure has been determined using NMR [3]. RNase Sa has proven to be an excellent model for various types of studies including structure-function relationship, mechanism of enzymatic reaction, protein-protein recognition, protein folding, flexibility and conformational stability of globular proteins.
For the better understanding the principles of conformational stability of proteins, single mutations in RNase Sa molecule were designed to remove a limited number of precisely defined hydrogen bonds and the stability of mutant proteins was measured [4,5,6,7]. Crystal structures of mutant proteins were determined in our laboratory at 1.0-1.7 Å resolution and the changes in hydrogen bonding were analyzed. It has been proved that i) intramolecular hydrogen bonds contribute substantially to the protein stability, ii) polar groups burial contribute to protein stability, iii) side chains on the surface of a protein that form intramolecular hydrogen bonds can make significant contributions to protein stability and iv) the effect of a single amino-acid mutation on conformational stability of protein highly depends on the location of the substitution and its environment in the structure.
Mutant proteins originally designed for the stability study have been analyzed also from the point of view of their catalytic properties, crystallizability and structural flexibility. Because of some difficulties at crystallization only eight structures have been published, yet. At present, there are additional six structures solved. Altogether, structures of fourteen mutants has been solved: two mutants of Asn 39, a residue conserved in the microbial T1 ribonuclease family (mutants N39S, N39D), three tyrosine to phenylalanine mutants (Y51F, Y80F, Y86F), four tryptophan mutants (D1W, Y55W, T76W, Y81W), three small polar and nonpolar group mutants (S24A, I71V, T95A) and two charged mutants (D79A, Q94K). Unexpected structural changes in the conformation of the surface loop have been observed in the case of N39S and N39D mutants. Two mutations, Q94K and T76W caused changes in the crystal packing, moreover, the main chain of the Q94K mutant is cleaved in the region of surface loop between Arg63 and Thr64. Structural flexibility has been also studied; the superposition of all mutant structures revealed the close similarity in in hydrophobic core and flexibility on the surface of the molecule, the picture is similar to the superposition of the structures solved by NMR method. Moreover, in some structures alternative conformations of main chain has been observed in various regions. The mutations influenced also the enzymatic activity in spite of the fact that none of mutated amino acid residues is directly involved into the substrate binding or cleaving. Asparagine 39 mutations caused changes in the conformation of the loop which forms a substrate binding pocket which resulted in the significant decrease of the activity to 20-2 %. Tyrosine 86 is positioned at the active site in the close proximity of the catalytic Glu54 and its mutation to phenylalanine led to decreasing the activity to 7 %. On the other hand, mutation of Ile71, which is positioned also very close to Glu54, to leucine, increased the enzyme activity 4 times. All the results of structure-function studies of RNase Sa mutants will be discussed in the presentation.
Acknowledgement. The work was supported by the Slovak Academy Research Grant Agency VEGA grant No. 2/0190/14. We acknowledge the EMBL c/o DESY, Hamburg for providing us with synchrotron source facilities.
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