Graduate Student, Caltech
New computer-based methods to avoid failed experiments thereby accelerating protein structure
Membrane proteins play a pivotal role in biology, representing a quarter of all proteomes and a majority of drug targets. While considerable effort has been focused on improving our functional understanding of this class, much of the investment has been hampered by the inability to obtain sufficient amounts of sample. Until now, there have been no broadly successful strategies for predicting and improving expression which means that each target requires an ad hoc adventure. Complex biological processes govern membrane protein expression; therefore, sequence characteristics that influence protein biogenesis are not simply additive. Many properties must be considered simultaneously in predicting the expression level of a protein.
We provide a first solution to the membrane protein expression problem by learning from published data to develop a statistical model that predicts the outcomes of expression trials across families, scales, and laboratories (all independent of the model’s training data). Given that the process of finding a target for large-scale expression is arduous, often requiring a long trial-and-error process that consumes significant financial and human resources, this work will have immediate applicability. The ability to study and engineer inaccessible membrane proteins becomes feasible with the use of our predictor. Furthermore, this work will enable others in developing new computational methods to assist in the experimental study of membrane proteins.
See more: http://shyam.saladi.org
Abstract: While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.
Pub.: 06 May '14, Pinned: 28 Jun '17
Abstract: Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs. lipidic) the protein encounters, and by the fact that most TM segments contain a substantial amount (~30%) of polar residues as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical - but sequentially different - peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous 'block' in the middle of the segment, or 'scrambled' throughout the sequence); and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima; and by extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum (ER). We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a "threshold hydrophilicity" is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.
Pub.: 14 Sep '16, Pinned: 28 Jun '17
Abstract: 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.
Pub.: 23 Mar '17, Pinned: 28 Jun '17