TY - JOUR

T1 - PH-dependent conformational changes of KcsA tetramer and monomer probed by Raman spectroscopy

AU - Kniggendorf, Ann Kathrin

AU - Schmidt, David

AU - Roth, Bernhard

AU - Plettenburg, Oliver

AU - Zeilinger, Carsten

N1 - Funding information: A.-K.K. received funding by the German Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education and Research) within the collaborative project OPTIMUS (13N13811) and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project ID 397827619. B.R. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy within the Cluster of Excellence PhoenixD (EXC 2122, Project ID 390833453). The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover.

PY - 2019/6/1

Y1 - 2019/6/1

N2 - KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm−1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm−1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.

AB - KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm−1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm−1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.

KW - 532 nm

KW - KcsA

KW - Liposome flux assay

KW - Monomer

KW - PH-dependent gating

KW - Raman spectroscopy

KW - Tetramer

UR - http://www.scopus.com/inward/record.url?scp=85067504415&partnerID=8YFLogxK

U2 - 10.3390/ijms20112736

DO - 10.3390/ijms20112736

M3 - Article

C2 - 31167355

AN - SCOPUS:85067504415

VL - 20

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 11

M1 - 2736

ER -