Zhang, Bin (2013) Sec-facilitated protein translocation and membrane integration. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:10172012-174905364
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The Sec translocon is a central component of the cellular machinery for targeting and delivering nascent proteins. Ubiquitous across all kingdoms of life, it is a protein-conducting channel that facilitates recognition of integral membrane protein domains and the establishment of integral membrane protein topology. Structural, biochemical, and biophysical studies have illuminated the role of the Sec translocon in both cotranslational and posttranslational protein targeting. In particular, quantitative assays have established the dependence of transmembrane domain (TM) stop-transfer efficiency and integral membrane protein topogenesis on the physicochemical properties of the translocon and protein nascent chain. These studies provide a valuable starting point for understanding the molecular regulation of the translocon and its sensitivity to mutations in protein sequence and external driving forces; however, complexities associated with the Sec machinery, including the role of collaborating molecular motors, the importance of large-scale conformational changes in the translocon, and the crowded molecular environment of the channel interior, obscure the mechanistic basis for many experimentally observed trends. My PhD research has focused on the development of a unified, mechanistic understanding of Sec-facilitated protein targeting.
Using both atomistic and coarse-grained molecular simulations, we have investigated the conformational landscape for the Sec translocon. We found that inclusion of a hydrophobic peptide substrate in the translocon stabilizes an open conformation of the lateral gate (LG) that is necessary for membrane integration, whereas inclusion of a hydrophilic peptide substrate favors only the closed LG conformation. We demonstrated that the translocon plug moiety adopts markedly different conformations in the channel, depending on whether the substrate peptide is hydrophobic or hydrophilic in character. Finally, we showed that the energetics of the translocon LG opening in the presence of the substrate peptides can be modeled in terms of the energetics of the peptide interface with the membrane. The manuscript associated with this study is published in PNAS, 107, 5399 (2010).
We further developed a novel computational protocol that combines nonequilibrium growth of the nascent protein with microsecond-timescale molecular dynamics trajectories. Analysis of multiple, long-timescale simulations elucidated molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon LG, large-lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrated the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups were shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (Ncyt/Cexo) integral membrane protein topology; and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer were shown to stabilize configurations that are consistent with the Type III (Nexo/Ccyt) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence. The manuscript associated with this study is published in J. Am. Chem. Soc., 134, 13700 (2012).
Finally, we introduced a coarse-grained modeling approach that spans the nanosecond to minute-timescale dynamics of cotranslational protein translocation. The method enabled direct simulation of both integral membrane protein topogenesis and TM stop-transfer efficiency. Simulations revealed multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrated that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon LG, and it was predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides new insight into the molecular regulation of the Sec translocon. The manuscript associated with this study is published in Cell Reports, in press.
This research has significantly enriched the mechanistic understanding of Sec-facilitated protein translocation and membrane integration with ample molecular details. The unifying picture that we propose establishes fundamental connections between previously disparate experimental studies, and it lays down the foundation for future verification and refinement.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Biophysics, Membrane Protein, Sec Translocon|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Awards:||The Herbert Newby McCoy Award, 2013|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||3 October 2012|
|Non-Caltech Author Email:||zhngbn (AT) outlook.com|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Bin Zhang|
|Deposited On:||14 Nov 2012 17:37|
|Last Modified:||02 Nov 2015 17:10|
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