TY - JOUR
T1 - Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins
AU - Hecht, Michael H.
AU - Sauer, Robert T.
N1 - Funding Information:
We are grateful to <Julian Sturtevant and Hillary Nelson for the communication of unpublished results: to (‘arl Pabo for the co-ordinat,es of the X-terminal domain: to Henry Shih and Martin Karplus for help with the molecular graphics; to Andrew Pakula for the cloning vector APpl; and to Fred Gimble for the anti-l repressor serum. This work was supported by National Institutes of Health grant AI-15706 and a grant from W. R,. Grace and Co.
PY - 1985/11/5
Y1 - 1985/11/5
N2 - We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to λ repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22 → Phe, Ser77 → Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35 → Leu, Gly48 → Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34 → Lys and Gly48 → Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.
AB - We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to λ repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22 → Phe, Ser77 → Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35 → Leu, Gly48 → Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34 → Lys and Gly48 → Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.
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U2 - 10.1016/0022-2836(85)90256-6
DO - 10.1016/0022-2836(85)90256-6
M3 - Article
C2 - 2934554
AN - SCOPUS:0022376431
VL - 186
SP - 53
EP - 63
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -