This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protein-conducting channel, more specifically known as the translocon (www.ks.uiuc.edu/Research/translocon/) or Sec complex, is an evolutionarily ancient protein complex that serves to help proteins cross or integrate into membranes (depending on whether they are to be soluble or membrane proteins) [139]. It exists in all branches of life and is found in the cytoplasmic membrane in bacteria and archaea and in the membrane of the endoplasmic reticulum in eukaryotes [140 142]. A passive channel by nature, it normally partners with other proteins that drive translocation of an unfolded polypeptide across the channel. For a common mode of translocation, co-translational translocation, this partner is the ribosome which feeds the nascent protein through the channel as it is being made [143, 144]. As a key step in protein targeting, translocation can be a deciding factor in the fate of proteins and even the cell as a whole. For example, poor recognition of the prion protein (PrP) can allow it to aggregate to lethal levels in the cell [145 147]. However, being able to enhance recognition and passage could benefit artificially created proteins such as insulin [148 150]. In 2004, the first available high resolution structure was released by our collaborator, Tom Rapoport; the protein was resolved at 3.5 Angstroms and obtained from the archaeon Methanococcus jannaschii [151]. Based on the new structure, the details of translocation began to come into focus; certain observed elements were proposed to have specific functions, such as a constrictive pore ring and a plug blocking the exit of the channel. It was also proposed that the monomer, and not a dimer or tetramer as hypothesized before, serves as the active channel. The Resource first approached this problem by building a system of the protein, a lipid bilayer large enough to surround it, and water and ions to best represent the channel s native environment, comprising in total a system of greater than 106,000 atoms. Using the program NAMD [44], channel crossing events for small polypeptide helices were simulated, with the system being allowed to relax both before and after [152]. With simulations totaling more than 40 ns, certain structural hypotheses were confirmed as well as new ones made. The pore ring was seen to behave as a tight yet flexible seal before, during, and after translocation, and residues other than the ones originally proposed [151] were suggested to play a role in maintaining the seal. The plug was also seen to behave in a manner expected, leaving the channel under the pressure of the pulled polypeptide but also returning to the pore when allowed to relax. Our combined results further confirmed the idea of the monomer as the active channel. Since this result still leaves open the question of why the protein oligomerizes in nature, we also simulated a back-to-back dimer of the channel, one of two currently proposed dimer forms, which required a noticeably larger system totaling 132,000 atoms. An equilibrium simulation of this structure using NAMD combined with analysis in VMD [49] demonstrated that the plug blocking each channel gained much greater flexibility than in simulations of the monomer. This observed interaction illustrates that two monomers may influence each other in beneficial ways not related to forming a larger channel. With new simulations, we are now examining both dimer forms, accurately constructed based on homology-modelled structures received from structural biologists in the field [153, 154]. Multiple simulation techniques will be used requiring many tens of nanoseconds in the hope of differentiating between the two dimer forms and realizing one as the more likely candidate for the in vivo complex.
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