De novo design of transmembrane β barrels.
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ABSTRACT:
SUBMITTER: Vorobieva AA
PROVIDER: S-EPMC8064278 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence. One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far. Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs. The method carefully attempts to avoid over-fitting the sparse experimental data. While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different. In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score. The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy). This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria. We found over 164 previously uncharacterized TMB proteins at high confidence. Database searches did not implicate any of these proteins with membranes. We challenge that the vast majority of our 164 predictions will eventually be verified experimentally. All proteome predictions and the PROFtmb prediction method are available at http://www.rostlab.org/ services/PROFtmb/.
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Project description:The biogenesis of transmembrane β-barrels (outer membrane proteins, or OMPs) is an elaborate multistep orchestration of the nascent polypeptide with translocases, barrel assembly machinery, and helper chaperone proteins. Several theories exist that describe the mechanism of chaperone-assisted OMP assembly in vivo and unassisted (spontaneous) folding in vitro. Structurally, OMPs of bacterial origin possess even-numbered strands, while mitochondrial β-barrels are even- and odd-stranded. Several underlying similarities between prokaryotic and eukaryotic β-barrels and their folding machinery are known; yet, the link in their evolutionary origin is unclear. While OMPs exhibit diversity in sequence and function, they share similar biophysical attributes and structure. Similarly, it is important to understand the intricate OMP assembly mechanism, particularly in eukaryotic β-barrels that have evolved to perform more complex functions. Here, we deliberate known facets of β-barrel evolution, folding, and stability, and attempt to highlight outstanding questions in β-barrel biogenesis and proteostasis.
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Project description:The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.
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Project description:Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.
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Project description:The regular arrangements of β-strands around a central axis in β-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of β-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.
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Project description:The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.
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Project description:HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with beta-propellor fold, ferredoxin-like fold, double psi barrels or (betaalpha)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a betaalphabetaalpha or betaalpha unit. HHrep can be accessed at http://hhrep.tuebingen.mpg.de.
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Project description:Transmembrane beta-barrels (TMBBs) belong to a special structural class of proteins predominately found in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. TMBBs are surface-exposed proteins that perform a variety of functions ranging from nutrient acquisition to osmotic regulation. These properties suggest that TMBBs have great potential for use in vaccine or drug therapy development. However, membrane proteins, such as TMBBs, are notoriously difficult to identify and characterize using traditional experimental approaches and current prediction methods are still unreliable.A prediction method based on the physicochemical properties of experimentally characterized TMBB structures was developed to predict TMBB-encoding genes from genomic databases. The Freeman-Wimley prediction algorithm developed in this study has an accuracy of 99% and MCC of 0.748 when using the most efficient prediction criteria, which is better than any previously published algorithm.The MS Windows-compatible application is available for download at http://www.tulane.edu/~biochem/WW/apps.html.
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Project description:[Figure: see text].
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