Early stage protein folding of the potassium channel Kcv

QH345 Biochemistry

Butiman, Chirapha ; Woolhead, C.

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: Kcv, transmembrane protein, potassium channel, early stage, secondary structure, folding, PEGylation assay, cross-linking assay.;   

Others  :  http://theses.gla.ac.uk/8712/1/2017Butimanmsc.pdf

来源: University of Glasgow

【 摘 要 】

Kcv is the shortest transmembrane protein across other voltage-and ligand-gated potassium channels, containing only 94 amino acids to form a functional K+ channel with two typical transmembrane domains (2TM).Current interest in cell biology raises questions about how this most primitive of proteins can function, given its very simple structure.To better understand the distinctive structure and function of Kcv, basic structural studies are necessary, especially in regard to how folding is vital to this protein’s conformation. Hence, we selected the Kcv as a small protein model to examine the early development of secondary structure during the synthesis within the ribosome throughout the tertiary structure and interaction of the nascent chain with the ribosomal protein L23 and signal recognition protein (SRP54). The research was carried out to monitor the early stages of protein folding in the 1st TM domain. The experimental strategy used the specific residues mutagenesis which involves these analysis techniques; using post-translation incorporated with the chemical modifications; PEGylation assay, cross-linking assay, and immunoprecipitation assay.The experimental processes was as follows: truncated ribosome nascent chain (RNCs) of interest had their lengths synthesised in the prokaryotic 30S in vitro transcription/ translation system, the N-terminus lengths were radio-labelled with [35S]met and eventually the post-translation products were topologically analysed using the chemical modifications;PEGylation (thiol PEG-MAL reagent), and cross-linked with two selective cross-linkers, either cysteine-cysteine cross-linker (1,6-bismaleimidoheane; BMH) or lysine-lysine cross-linker (bis-(sulfosuccinimidyl)suberate;(BS3), and subsequently encompassing comprehensive analysis of the cross-linked products using immunoprecipitation (IP) with two desired antibodies; L23 and the signal recognition particle (SRP), respectively. The samples were resolved on SDS-PAGE gels with the autoradiography.The PEGylation image successfully presented the clear PEGylated bands from 50-70aa when employing the M15L-Cysteine (incorporating L8C with a single methionine replacement) whereas the cross-linking assay using BS3 associated with immunoprecipitation assay presented the clearest bands from 50-70 aa when using the three single lysines (K29I/K46/47M) incorporated with the single three methionine replacement (M15/23/26L) called ‘M15/23/26L-Lysine’.This project proposes that the small 2TM protein, Kcv, begins to form as a folded structure inside the ribosome tunnel. Additionally, data from this project suggests that the helical 1st transmembrane region of the Kcv could be first compacted in the ribosomal exit tunnel before attaching to the targeting pathway. Based on the PEGylated band from the PEGylation assay, the nascent chain is exported from the ribosome tunnel at approximately 50 aa (~30aa contributed inside the ribosome; non-PEGylated), while consequent timing of the nascent chains interaction with L23 and SRP, which have been cross-linked and precipitated with the same PEGylation profile, shows that the interaction begins to be manifested from 50aa. These results agree with previous data for the 2TM protein (F0c) but are at odds with our labs analysis of larger 7TM protein (GPCR GPR35). Furthermore, the interaction between nascent chains, L23, and SRP54 in the ribosome exit tunnel are able to demonstrate a responsibility outline principle to the further cellular targeting pathway of Kcv, a small membrane protein which is inserted into the plasma membrane, which is the final destination.