Transmembrane and extramembrane contributions to membrane protein thermal stability: studies with the NaChBac sodium channel.
The thermal stabilities of the extramembranous and transmembranous regions of the bacterial voltage-gated sodium channel NaChBac have been characterised using thermal-melt synchrotron radiation circular dichroism (SRCD) spectroscopy. A series of constructs, ranging from the full-length protein containing both the C-terminal cytoplasmic and the transmembranous domains, to proteins with decreasing amounts of the cytoplasmic domain, were examined in order to separately define the roles of these two types of domains in the stability and processes of unfolding of a membrane protein. The sensitivity of the SRCD measurements over a wide range of wavelengths and temperatures has meant that subtle but reproducible conformational changes could be detected with accuracy. The residues in the C-terminal extramembranous domain were highly susceptible to thermal denaturation, but for the most part the transmembrane residues were not thermally-labile and retained their helical character even at very elevated temperatures. The process of thermal unfolding involved an initial irreversible unfolding of the highly labile distal extramembranous C-terminal helical region, which was accompanied by a reversible unfolding of a small number of helical residues in the transmembrane domain. This was then followed by the irreversible unfolding of a limited number of additional transmembrane helical residues at greatly elevated temperatures. Hence this study has been able to determine the different contributions and roles of the transmembrane and extramembrane residues in the processes of thermal denaturation of this multipass integral membrane protein.
Department of Crystallography, University of London, London, UK.
Andrew M
AM
Powl
Andrew J
AJ
Miles
B A
BA
Wallace
D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D006358:Hot Temperature; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000737:chemistry*
D001426:Bacterial Proteins; C443666:NaChBac protein, bacteria; D015222:Sodium Channels