Computational protein design
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ix, 682 s. : il., tab.
- Klíčová slova
- Klonování, Proteiny,
- MeSH
- klonování DNA MeSH
- molekulární biologie MeSH
- proteiny MeSH
- vazba proteinů fyziologie MeSH
- Publikační typ
- laboratorní příručky MeSH
Enzymes are the natural catalysts that execute biochemical reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial production of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technological advances have greatly facilitated this process by providing the experimental approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous laboratory work and economic resources. Theoretical methods provide predictions and represent an attractive complement to such experiments by waiving their inherent costs. Computational techniques can be used to engineer enzymatic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, solubility, and flexibility. Theoretical approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.
Protein engineering strategies aimed at constructing enzymes with novel or improved activities, specificities, and stabilities greatly benefit from in silico methods. Computational methods can be principally grouped into three main categories: bioinformatics; molecular modelling; and de novo design. Particularly de novo protein design is experiencing rapid development, resulting in more robust and reliable predictions. A recent trend in the field is to combine several computational approaches in an interactive manner and to complement them with structural analysis and directed evolution. A detailed investigation of designed catalysts provides valuable information on the structural basis of molecular recognition, biochemical catalysis, and natural protein evolution.
- MeSH
- enzymy genetika MeSH
- lidé MeSH
- molekulární modely MeSH
- mutace MeSH
- proteinové inženýrství metody MeSH
- stabilita enzymů MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.
- MeSH
- bodová mutace genetika fyziologie MeSH
- databáze genetické MeSH
- lyasy chemie genetika metabolismus MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- proteinové inženýrství metody MeSH
- stabilita enzymů genetika MeSH
- teplota MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
β-sheet proteins carry out critical functions in biology, and hence are attractive scaffolds for computational protein design. Despite this potential, de novo design of all-β-sheet proteins from first principles lags far behind the design of all-α or mixed-αβ domains owing to their non-local nature and the tendency of exposed β-strand edges to aggregate. Through study of loops connecting unpaired β-strands (β-arches), we have identified a series of structural relationships between loop geometry, side chain directionality and β-strand length that arise from hydrogen bonding and packing constraints on regular β-sheet structures. We use these rules to de novo design jellyroll structures with double-stranded β-helices formed by eight antiparallel β-strands. The nuclear magnetic resonance structure of a hyperthermostable design closely matched the computational model, demonstrating accurate control over the β-sheet structure and loop geometry. Our results open the door to the design of a broad range of non-local β-sheet protein structures.
- MeSH
- konformace proteinů, beta-řetězec MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- počítačová simulace MeSH
- proteinové inženýrství metody MeSH
- proteiny chemie genetika MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- alosterická regulace * MeSH
- alosterické místo * MeSH
- benzodiazepiny farmakokinetika farmakologie MeSH
- farmaceutická chemie MeSH
- farmakokinetika MeSH
- kalcimimetika farmakokinetika farmakologie MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- neurotransmiterové látky farmakokinetika farmakologie MeSH
- racionální návrh léčiv MeSH
- receptory GABA-A * fyziologie MeSH
- receptory spřažené s G-proteiny fyziologie MeSH
- sirtuin 1 farmakokinetika farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.
- MeSH
- automatizace MeSH
- biokatalýza MeSH
- databáze proteinů MeSH
- internet * MeSH
- molekulární evoluce MeSH
- molekulární modely MeSH
- mutace * MeSH
- mutageneze cílená metody MeSH
- peptidová knihovna * MeSH
- proteiny chemie genetika MeSH
- software * MeSH
- stabilita proteinů MeSH
- substituce aminokyselin MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
