The role of a trans-proline in the folding mechanism of ribonuclease T1.
Protein folding is often retarded by the cis reversible trans isomerizations of prolyl peptide bonds both in vitro and in vivo. An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolines) by comparing the wild-type protein with the Pro73-->Val variant. The Pro-->Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding. In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged. trans-Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines. The cis-proline residues dominate the kinetics of refolding, because almost all slow-folding molecules contain the respective incorrect (trans) isomers, and because trans-->cis isomerizations are slower than cis-->trans isomerizations. The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this proline is non-essential for folding in the sense that its cis reversible trans isomerization is energetically uncoupled from conformational folding.
Schindler
T
T
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Mayr
L M
LM
Landt
O
O
Hahn
U
U
Schmid
F X
FX
D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013816:Thermodynamics
D011994:Recombinant Proteins; D011392:Proline; D006163:Ribonuclease T1