TY - JOUR
T1 - Solution NMR and CD spectroscopy of an intrinsically disordered, peripheral membrane protein
T2 - Evaluation of aqueous and membrane-mimetic solvent conditions for studying the conformational adaptability of the 18.5 kDa isoform of myelin basic protein (MBP)
AU - Libich, David S.
AU - Harauz, George
N1 - Funding Information:
Acknowledgments This work was supported by the Canadian Institutes of Health Research (Operating Grant MOP 74468), and the Natural Sciences and Engineering Research Council of Canada (Operating Grant RG121541). DSL was the recipient of an Ontario Graduate Scholarship. The University of Guelph NMR Centre bene-Wted from funding by the Canada Foundation for Innovation, and the Ontario Innovation Trust. The authors are grateful to Dr. Vladimir Bamm, Ms. Valerie Robertson (Guelph NMR Center), Dr. Martine Monette (Bruker BioSpin Canada), and Dr. Vladimir Ladizhansky (Physics, Guelph) for many helpful discussions, advice, and support.
PY - 2008/7
Y1 - 2008/7
N2 - The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy (1H- 15N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d2 in H2O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.
AB - The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy (1H- 15N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d2 in H2O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.
KW - Chemical shift index
KW - Circular dichroism
KW - HSQC spectrum
KW - Intrinsically disordered protein
KW - Myelin basic protein
KW - Solution NMR spectroscopy
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U2 - 10.1007/s00249-008-0334-8
DO - 10.1007/s00249-008-0334-8
M3 - Article
C2 - 18449534
AN - SCOPUS:45849135972
SN - 0175-7571
VL - 37
SP - 1015
EP - 1029
JO - European Biophysics Journal
JF - European Biophysics Journal
IS - 6
ER -