Connie Wang

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.

Allosteric Response and Substrate Sensitivity in Peptide Binding of the Signal Recognition Particle

Background: The SRP is a central component of the co-translational protein targeting pathway. Results: Long timescale computer simulations reveal shifts in the SRP conformational distribution upon nascent protein binding. Conclusion: The binding-induced conformational shifts correlate with the experimentally observed efficiency of protein targeting. Significance: The work provides new insight into the mechanism by which SRP allostery regulates the fidelity of protein targeting. We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway.

Don't look at the eyes: Live interaction reveals strong eye avoidance behavior in autism

Atypical social gaze is commonly observed in individuals with autism (ASD) in real-world and clinical settings. Laboratory tasks using social stimuli have shown reduced gaze to face and eyes and reduced social orienting in high-functioning adults compared to neurotypical (NT) controls, although differences were often marginal, perhaps due to static stimuli or non-interactive tasks. In this study, we investigated gaze during live, naturalistic interactions between pairs of participants conversing freely about their interests, while gaze and video were recorded for both. Results from 8 NT and 7 ASD participants revealed distinct gaze patterns, distinguishing the groups. All NT participants displayed a consistent pattern of high gaze frequency and duration (mean=54%) to the eyes and low gaze to the mouth (mean=1%). ASD participants showed significantly lower gaze frequency (mean=10%, p< 0.00000001) and duration (mean=7%, p< 0.000001) to the eyes, with higher frequency (mean=33%, p< 0.02) and duration (mean=39%, p< 0.02) to the mouth, and no difference for the face (NT mean=77%, ASD mean=72%, n.s.). Only NTs showed a significant preference for the left eye in frequency (p< 0.05) and duration (p< 0.04). Mouth gaze split ASD participants into subgroups of high (N=4) or low (N=3) frequency, but long mouth fixations characterized ASD overall (640 ms) and distinguished (p< 0.01) from short fixations (210 ms) in NT. Together, these results show that live, interactive experiments can detect striking differences in social gaze between NT and ASD groups. The NT pattern is defined by high eye contact with occasional, passing glances at the mouth, while ASD shows a strong, spontaneous tendency to avoid the eyes and prolonged fixations to the mouth. Diversion of gaze to the mouth or other face regions (e.g. nose, cheeks, forehead) suggests a compensatory mechanism for eye avoidance that allows face gaze without direct eye-to-eye contact in ASD. Meeting abstract presented at VSS 2015.

ProtaBank: A repository for protein design and engineering data

We present ProtaBank, a repository for storing, querying, analyzing, and sharing protein design and engineering data in an actively maintained and updated database. ProtaBank provides a format to describe and compare all types of protein mutational data, spanning a wide range of properties and techniques. It features a user‐friendly web interface and programming layer that streamlines data deposition and allows for batch input and queries. The database schema design incorporates a standard format for reporting protein sequences and experimental data that facilitates comparison of results across different data sets. A suite of analysis and visualization tools are provided to facilitate discovery, to guide future designs, and to benchmark and train new predictive tools and algorithms. ProtaBank will provide a valuable resource to the protein engineering community by storing and safeguarding newly generated data, allowing for fast searching and identification of relevant data from the existing literature, and exploring correlations between disparate data sets. ProtaBank invites researchers to contribute data to the database to make it accessible for search and analysis. ProtaBank is available at https://protabank.org.

Inversion of Signal Sequence Topology during Membrane Integration

In many cases, the topology of membrane proteins is established during co-translational membrane integration. This process involves the Sec translocon, a heterotrimeric protein-conducting channel that allows for both the translocation of secreted domains across the membrane through the central pore and the integration of membrane domains directly into the lipid bilayer through a lateral opening. For many proteins, the N-terminus is retained on the cytosolic side of the membrane and forms an inverted (type II) topology that threads the C-terminus through the channel. This inverted topology is hypothesized to involve a head-first intermediate which then undergoes a step-wise inversion process to its final topology. We use a newly developed coarse-grained model to simulate the integration of the signal sequence during the elongation of the nascent chain on the minute-long timescales that are relevant to the biological process. This coarse-grained simulation method enables direct comparisons to experimentally measured energetics of ribosome-nascent chain to translocon interactions. We observe a series of pulling and pushing forces on the ribosome-nascent chain as translation proceeds and identify a head-first intermediate whose inversion is driven by the entropic confinement of nascent chain residues in the ribosome-translocon junction.