Probing the unfolding region of ribonuclease A by site-directed mutagenesis.
Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.
Department of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Jens
J
Köditz
Renate
R
Ulbrich-Hofmann
Ulrich
U
Arnold
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013820:Thermolysin / Q000737:chemistry; D014170:Transformation, Genetic
D000596:Amino Acids; D009841:Oligonucleotides; D011994:Recombinant Proteins; D012997:Solvents; D012259:Ribonuclease, Pancreatic; D013820:Thermolysin; D019791:Guanidine