Development of a Mechanism and Structure-Guided Methodology for De Novo Enzyme Design
开发从头酶设计的机制和结构引导方法
基本信息
- 批准号:10457838
- 负责人:
- 金额:$ 1.79万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-07-01 至 2022-09-17
- 项目状态:已结题
- 来源:
- 关键词:AccelerationActive SitesAddressAffinityAgreementAntibiotic ResistanceBenchmarkingBindingBinding ProteinsBinding SitesBioinformaticsBiological ModelsBiophysicsCalorimetryCatalysisChemicalsChemistryCircular DichroismComplementComplexDevelopmentDirected Molecular EvolutionDockingEnzymesEvolutionExhibitsFoundationsHealthHumanHydrogen BondingHydrolysisHydrophobicityIn VitroIndividualInvestigationKineticsLactamaseLactamsLigand BindingLigandsMaintenanceMeasuresMetalsMethodologyMethodsModelingMonobactamsNMR SpectroscopyOpticsPositioning AttributePropertyProtein EngineeringProteinsProtonsReactionRoleScaffolding ProteinSiteSpecificitySpectrum AnalysisStructureStructure-Activity RelationshipSubstrate SpecificityTertiary Protein StructureTestingTherapeuticWorkX-Ray Crystallographyanalogaqueousbasebeta-Lactamasebeta-Lactamsbioinformatics toolbiological systemsbiophysical techniqueschemical reactiondesignfunctional grouphigh throughput screeningimprovedin silicoinsightiterative designnovelprediction algorithmprotein complexprotein functionprotein structureprotein structure functionrational designreaction ratesimulationsmall molecule
项目摘要
Project Summary/Abstract
Naturally enzymes are characterized by their ability to accelerate chemical reactions by orders of
magnitude and with far greater specificity than those observed in aqueous solution. However, these catalytic
properties have yet to be realized in synthetic chemical or biological systems. While there have been many
recent advances in the de novo design of proteins, achieving the exquisite control of individual atoms and
functional groups necessary for enzyme catalysis remains a long-standing challenge and has relied on directed
evolution and high-throughput screening to improve upon designs. The first principles design of an active site
capable of substrate binding and catalysis with rates and affinities similar to those found in natural enzymes
would represent a breakthrough in our understanding of structure-function relationships and the origins of protein
(dys)function. A recent advance in the de novo design of small-molecule-binding proteins demonstrates the
ability to position non-covalent interactions, such as hydrogen bonds from a protein to ligand functional groups,
with sub-Å accuracy. Based on this methodology, it’s hypothesized that if non-covalent interactions can be
rationally designed for binding, then they can be directed towards achieving de novo enzyme design through
preferential TS-stabilization to achieve fast reaction rates. In order to test this hypothesis, enzymes capable of
the model Kemp elimination reaction will be de novo designed using a bottom-up approach to install a general
base and tune its reactivity, assemble an active site capable of substrate- and TS-analog-binding, and develop
a negative design strategy for preferential TS-stabilization. These fundamental insights will be directed towards
the first de novo metallo-β-lactamase, a model system for studying antibiotic resistance and protein evolution
with human health implications. Catalytic turnover of the large and highly polar β-lactam antibiotics provides a
sensitive test of our ability to design non-covalent interactions. This will be achieved by expanding upon the size,
asymmetry, and topology of designable protein scaffolds using a bioinformatic and function-guided design
strategy for multi-domain proteins, de novo design of a Zn2+- and substrate-binding site, and development of de
novo methods for preferential transition-state stabilization. The proposed investigations will be achieved using
both computational and experimental methods, starting from in silico approaches to benchmark the folding and
binding of reaction intermediates in designed proteins. Promising designs will then be expressed, purified, and
characterized in terms of their binding affinity and catalytic rate constants using isothermal calorimetry and optical
spectroscopies. These functional studies will be complemented by structural characterization using X-ray
crystallography and NMR spectroscopy to confirm the accuracy of the design methodology. Successful de novo
design of functional enzymes would represent a breakthrough in our understanding of structure-function
relationships and the role of non-covalent interaction in complex protein function.
项目总结/文摘
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Samuel H. Schneider其他文献
The Physical Origins of Enzyme Evolution: Correlating the Active Site Electric Fields of Antibiotic Resistance along Evolutionary Trajectories in TEM β-Lactamases
- DOI:
10.1016/j.bpj.2017.11.1117 - 发表时间:
2018-02-02 - 期刊:
- 影响因子:
- 作者:
Samuel H. Schneider;Jacek A. Kozuch;Steven G. Boxer - 通讯作者:
Steven G. Boxer
Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.
缓慢的扩散和排除体积都有助于大分子拥挤对乙醇脱氢酶稳态动力学的影响。
- DOI:
- 发表时间:
2015 - 期刊:
- 影响因子:2.9
- 作者:
Samuel H. Schneider;Schuyler Lockwood;Dominique I Hargreaves;David J. Slade;M. LoConte;Bridget E Logan;Erin E McLaughlin;M. Conroy;Kristin M. Slade - 通讯作者:
Kristin M. Slade
Tradeoffs of electrostatics and chemical positioning in the evolution of antibiotic resistance in TEM β-lactamases
- DOI:
10.1016/j.bpj.2021.11.1024 - 发表时间:
2022-02-11 - 期刊:
- 影响因子:
- 作者:
Steven G. Boxer;Samuel H. Schneider;Jacek A. Kozuch - 通讯作者:
Jacek A. Kozuch
De novo design of a β-lactamase in a modular helical bundle protein scaffold
- DOI:
10.1016/j.bpj.2021.11.2477 - 发表时间:
2022-02-11 - 期刊:
- 影响因子:
- 作者:
Samuel H. Schneider;William DeGrado - 通讯作者:
William DeGrado
Vibrational Stark Effects for Diverse Carbonyl Probes Applied to the Re-Interpretation of IR and Raman Data in Terms of Electric Fields at Enzyme Active Sites
不同羰基探针的振动斯塔克效应应用于根据酶活性位点的电场重新解释红外和拉曼数据
- DOI:
- 发表时间:
2016 - 期刊:
- 影响因子:0
- 作者:
Samuel H. Schneider;S. Boxer - 通讯作者:
S. Boxer
Samuel H. Schneider的其他文献
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{{ truncateString('Samuel H. Schneider', 18)}}的其他基金
Development of a Mechanism and Structure-Guided Methodology for De Novo Enzyme Design
开发从头酶设计的机制和结构引导方法
- 批准号:
10231920 - 财政年份:2021
- 资助金额:
$ 1.79万 - 项目类别:
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