Premium
Trapping the fast‐refolding state of ribonuclease A at subzero temperatures
Author(s) -
Fink Anthony L.,
Anderson William D.,
Antonino Lisa
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80810-x
Subject(s) - ribonuclease , trapping , chemistry , state (computer science) , biophysics , biochemistry , computer science , biology , rna , algorithm , ecology , gene
Unfolded ribonuclease A consists of a mixture of fast‐ and slow‐refolding species. It is generally accepted that the slow‐refolding states arise from isomerization of proline residues. We show that unfolding at subzero temperatures may be used to trap the fast‐refolding species U f , since the rate of proline isomerization slows down at a much faster rate than protein unfolding. The unfolding was carried out in 5 M guanidine hydrochloride; at −15°C the protein unfolding process is complete within 30 s and under these conditions there is < 1.5% proline isomerization. By using ribonuclease in which Tyr‐115 was nitrated it was possible to rule out significant isomerization of Pro‐114 in the observed slow‐unfolding step.