GMMA Can Stabilize Proteins Across Different Functional Constraints
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GMMA Can Stabilize Proteins Across Different Functional Constraints. / Daffern, Nicolas; Johansson, Kristoffer E.; Baumer, Zachary T.; Robertson, Nicholas R.; Woojuh, Janty; Bedewitz, Matthew A.; Davis, Zoë; Wheeldon, Ian; Cutler, Sean R.; Lindorff-Larsen, Kresten; Whitehead, Timothy A.
In: Journal of Molecular Biology, Vol. 436, No. 11, 168586, 2024.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - GMMA Can Stabilize Proteins Across Different Functional Constraints
AU - Daffern, Nicolas
AU - Johansson, Kristoffer E.
AU - Baumer, Zachary T.
AU - Robertson, Nicholas R.
AU - Woojuh, Janty
AU - Bedewitz, Matthew A.
AU - Davis, Zoë
AU - Wheeldon, Ian
AU - Cutler, Sean R.
AU - Lindorff-Larsen, Kresten
AU - Whitehead, Timothy A.
N1 - Publisher Copyright: © 2024 Elsevier Ltd
PY - 2024
Y1 - 2024
N2 - Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2–6 °C in thermal inactivation temperature and no decrease in functionality.
AB - Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2–6 °C in thermal inactivation temperature and no decrease in functionality.
KW - Computational Design
KW - Deep Sequencing
KW - GMMA
KW - Protein Engineering
KW - Protein Thermostability
U2 - 10.1016/j.jmb.2024.168586
DO - 10.1016/j.jmb.2024.168586
M3 - Journal article
C2 - 38663544
AN - SCOPUS:85191955427
VL - 436
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 11
M1 - 168586
ER -
ID: 391677846