Contribution of a Conserved Asparagine to the Conformational Stability of Ribonucleases Sa, Ba, and T1^,

Abstract

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.

Amino Acid Contribution to Protein Solubility: Asp, Glu, and Ser Contribute more Favorably than the other Hydrophilic Amino Acids in RNase Sa

“…These mutations were modeled using Swiss-PDBViewer 66 and the percent burial of the carboxyl oxygens were calculated using pfis 37 (Table 7). Substitution of these glutamines with glutamic acid would have allowed the carboxyl oxygens to be more exposed to solvent.…”

“…Position 76 in RNase Sa and neighboring turn residues (Ala75, Gln77, and Glu78). The side chain of Thr76 is 2.5% hyper-exposed to solvent as determined by pfis 37 . The figure was generated using the Swiss-Pdb Viewer program 66 .…”

“…Measurements at high positive net charge, low net charge, and high negative net charge have also been made. A threonine residue is located at position 76 of wild-type RNase Sa, and it is 2.5% hyperexposed to solvent compared to a model Ala-X-Ala peptide as determined by the program pfis 37 . Therefore, every amino acid introduced at position 76 is probably completely solvent exposed, thereby allowing comparison of the contributions to protein solubility of the entire side-chains of all twenty amino acids.…”

The Contribution of Polar Group Burial to Protein Stability Is Strongly Context-dependent

“…Thermal Denaturation-The thermal denaturation of RNase Sa and all of the single mutants studied to date is reversible and closely approaches a two-state folding mechanism (15,26,28). Thermal denaturation curves were determined and analyzed as described above, and the results are presented in Table II.…”

“…We have previously compared the stability of RNase Sa and two close relatives, RNase Sa2 and RNase Sa3, and determined the pH and salt dependence of their stability (25). We have shown that a conserved Asn residue (26) and the Tyr -OH groups (15) make large contributions to the stability of RNase Sa. We have studied the contribution of charge-charge interactions to the stability of RNase Sa (27).…”