25 research outputs found
Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon
An important aspect of cellular function is the correct targeting and delivery of newly synthesized proteins. Central to this task is the machinery of the Sec translocon, a transmembrane channel that is involved in both the translocation of nascent proteins across cell membranes and the integration of proteins into the membrane. Considerable experimental and computational effort has focused on the Sec translocon and its role in nascent protein biosynthesis, including the correct folding and expression of integral membrane proteins. However, the use of molecular simulation methods to explore Sec-facilitated protein biosynthesis is hindered by the large system sizes and long (i.e., minute) timescales involved. In this work, we describe the development and application of a coarse-grained simulation approach that addresses these challenges and allows for direct comparison with both in vivo and in vitro experiments. The method reproduces a wide range of experimental observations, providing new insights into the underlying molecular mechanisms, predictions for new experiments, and a strategy for the rational enhancement of membrane protein expression levels
Force transduction creates long-ranged coupling in ribosomes stalled by arrest peptides
Force-sensitive arrest peptides regulate protein biosynthesis by stalling the ribosome as they are translated. Synthesis can be resumed when the nascent arrest peptide experiences a pulling force of sufficient magnitude to break the stall. Efficient stalling is dependent on the specific identity of a large number of amino acids, including amino acids which are tens of angstroms away from the peptidyl transferase center (PTC). The mechanism of force-induced restart and the role of these essential amino acids far from the PTC is currently unknown. We use hundreds of independent molecular dynamics trajectories spanning over 120 μs in combination with kinetic analysis to characterize the barriers along the force-induced restarting pathway for the arrest peptide SecM. We find that the essential amino acids far from the PTC play a major role in controlling the transduction of applied force. In successive states along the stall-breaking pathway, the applied force propagates up the nascent chain until it reaches the C-terminus of SecM and the PTC, inducing conformational changes that allow for restart of translation. A similar mechanism of force propagation through multiple states is observed in the VemP stall-breaking pathway, but secondary structure in VemP allows for heterogeneity in the order of transitions through intermediate states. Results from both arrest peptides explain how residues that are tens of angstroms away from the catalytic center of the ribosome impact stalling efficiency by mediating the response to an applied force and shielding the amino acids responsible for maintaining the stalled state of the PTC
Improving membrane protein expression by optimizing integration efficiency
The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies
Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration
We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency
Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon
During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide (AP) experiments and coarse-grained molecular dynamics (CGMD) to measure and elucidate the molecular origins of forces that are exerted on NCs during co-translational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a read-out of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis
Water Dynamics Around Proteins: T- and R-States of Hemoglobin and Melittin
The water dynamics, as characterized by the local hydrophobicity (LH), is
investigated for tetrameric hemoglobin and dimeric melittin. For the T0 to R0
transition in Hb it is found that LH provides additional molecular-level
insight into the Perutz mechanism, i.e., the breaking and formation of salt
bridges at the alpha1 / beta2 and alpha2 / beta1 interface is accompanied by
changes in LH. For Hb in cubic water boxes with 90 Aengstroem and 120
Aengstroem edge length it is observed that following a decrease in LH as a
consequence of reduced water density or change of water orientation at the
protein/water interface the alpha / beta interfaces are destabilized; this is a
hallmark of the Perutz stereochemical model for the T to R transition in Hb.
The present work thus provides a dynamical view of the classical structural
model relevant to the molecular foundations of Hb function. For dimeric
melittin, earlier results by Cheng and Rossky (Nature, 1998, 392, 696-699) are
confirmed and interpreted on the basis of LH from simulations in which the
protein structure is frozen. For the flexible melittin dimer the changes in the
local hydration can be as much as 30 % than for the rigid dimer, reflecting the
fact that protein and water dynamics are coupled
Improving membrane protein expression by optimizing integration efficiency
The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies
A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency
Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design
Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon
During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide (AP) experiments and coarse-grained molecular dynamics (CGMD) to measure and elucidate the molecular origins of forces that are exerted on NCs during co-translational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a read-out of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis
A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency
Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design