tailieunhanh - Báo cáo khoa học: Probing the unfolding region of ribonuclease A by site-directed mutagenesis

Ribonuclease A contains two exposed loop regions, around Ala20 andAsn34. Only the loop aroundAla20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacentb-sheet aroundThr45) is the first region of the ribonuclease A molecule that becomes sus-ceptible to thermolysin and trypsin under unfolding condi-tions. 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 | Eur. J. Biochem. 271 4147-4156 2004 FEBS 2004 doi Probing the unfolding region of ribonuclease A by site-directed mutagenesis Jens Koditz Renate Ulbrich-Hofmann and Ulrich Arnold Department of Biochemistry and Biotechnology Martin-Luther University Halle-Wittenberg Halle Germany 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 b-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 orN34D 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 AG 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 .