TY - JOUR
T1 - Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination
AU - Shibata, Yoko
AU - Gvozdenovic-Jeremic, Jelena
AU - Love, James
AU - Kloss, Brian
AU - White, Jim F.
AU - Grisshammer, Reinhard
AU - Tate, Christopher G.
N1 - Funding Information:
This research was supported by a joint grant from Pfizer Global Research and Development and the MRCT Development Gap Fund in addition to core funding from the UK Medical Research Council MRC U105197215 (YS, CGT) and the Intramural Research Program of the National Institutes of Health, National Institute of Neurological Disorders and Stroke (JFW, JG-J, RG). The Protein Production Facility of the New York Consortium on Membrane Protein Structure was supported by the National Institutes of Health grant U54GM075026 (JL, BK). DNA sequence analysis was performed by the National Institute of Neurological Disorders and Stroke DNA Sequencing Facility.
PY - 2013/4
Y1 - 2013/4
N2 - Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.
AB - Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.
KW - Membrane protein
KW - Neurotensin receptor
KW - Structure
KW - Thermostability
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U2 - 10.1016/j.bbamem.2013.01.008
DO - 10.1016/j.bbamem.2013.01.008
M3 - Article
C2 - 23337476
AN - SCOPUS:84873714761
SN - 0005-2736
VL - 1828
SP - 1293
EP - 1301
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 4
ER -