TY - JOUR
T1 - Study of Small-Molecule-Membrane Protein Binding Kinetics with Nanodisc and Charge-Sensitive Optical Detection
AU - Ma, Guangzhong
AU - Guan, Yan
AU - Wang, Shaopeng
AU - Xu, Han
AU - Tao, Nongjian
N1 - Funding Information:
We thank the Innovative Molecular Analysis Technologies (IMAT) program from National Cancer Institute, National Institutes of Health (1R21CA173205), and Amgen Inc. for financial support.
PY - 2016/2/16
Y1 - 2016/2/16
N2 - Nanodisc technology provides membrane proteins with a nativelike lipid bilayer and much-needed solubility and enables in vitro quantification of membrane protein binding with ligands. However, it has been a challenge to measure interaction between small-molecule ligands and nanodisc-encapsulated membrane proteins, because the responses of traditional mass-based detection methods scale with the mass of the ligands. We have developed a charge-sensitive optical detection (CSOD) method for label-free measurement of the binding kinetics of low molecular mass ligands with nanodisc-encapsulated membrane proteins. This microplate-compatible method is sensitive to the charge instead of the mass of a ligand and is able to measure both large and small molecules in a potentially high-throughput format. Using CSOD, we measured the binding kinetics between peptide and small-molecule ligands and a nanodisc-encapsulated potassium ion channel protein, KcsA-Kv1.3. Both association and dissociation rate constants for these ligands are obtained for the first time. The CSOD results were validated by the consistency of the values with reported binding affinities. In addition, we found that CSOD can tolerate up to 3.9% dimethyl sulfoxide (DMSO) and up to 10% serum, which shows its compatibility with realistic sample conditions.
AB - Nanodisc technology provides membrane proteins with a nativelike lipid bilayer and much-needed solubility and enables in vitro quantification of membrane protein binding with ligands. However, it has been a challenge to measure interaction between small-molecule ligands and nanodisc-encapsulated membrane proteins, because the responses of traditional mass-based detection methods scale with the mass of the ligands. We have developed a charge-sensitive optical detection (CSOD) method for label-free measurement of the binding kinetics of low molecular mass ligands with nanodisc-encapsulated membrane proteins. This microplate-compatible method is sensitive to the charge instead of the mass of a ligand and is able to measure both large and small molecules in a potentially high-throughput format. Using CSOD, we measured the binding kinetics between peptide and small-molecule ligands and a nanodisc-encapsulated potassium ion channel protein, KcsA-Kv1.3. Both association and dissociation rate constants for these ligands are obtained for the first time. The CSOD results were validated by the consistency of the values with reported binding affinities. In addition, we found that CSOD can tolerate up to 3.9% dimethyl sulfoxide (DMSO) and up to 10% serum, which shows its compatibility with realistic sample conditions.
UR - http://www.scopus.com/inward/record.url?scp=84958205897&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84958205897&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.5b04366
DO - 10.1021/acs.analchem.5b04366
M3 - Article
C2 - 26752355
AN - SCOPUS:84958205897
VL - 88
SP - 2375
EP - 2379
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 4
ER -