Monitoring and Tuning a Gas-Binding Heme Protein with Unnatural Amino Acids
Monitoring and Tuning a Gas-Binding Heme Protein with Unnatural Amino Acids
批准号:
9231766
负责人:
Christine Marie Phillips Piro
金额:
$37.17万
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-09-15 至 2020-08-31
关键词:
AffinityAmberAmino AcidsBacteriaBindingBinding ProteinsBiochemicalBlood SubstitutesBlood VesselsCellsCodon NucleotidesDevelopmentDiseaseDissociationDistalEngineeringEnvironmentEukaryotic CellGasesGoalsHealthHemeHumanHydrogen BondingHypoxiaLasersLearningLigand BindingLigaseLocationMapsMethodologyMinorMonitorNitric OxideNitrilesOutcomes ResearchOxygenPhenylalaninePlayProkaryotic CellsPropertyProteinsReporterReportingResearchRoentgen RaysRoleShewanellaSignal TransductionSignal Transduction PathwaySiteSoluble Guanylate CyclaseSpectroscopy, Fourier Transform InfraredSpectrum AnalysisStructureSystemTherapeutic AgentsThermoanaerobacterTyrosineWorkX-Ray Crystallographybiophysical techniquesdesignflash photolysisfunctional groupheme-binding proteinimprovedinsightinterestmemberminimally invasivemutantprotein functionprotein structuresensortherapeutic proteintherapy developmenttoolunnatural amino acids
中文摘要
项目摘要。
血红素一氧化氮和/或氧结合(H-NOX)蛋白形成一类涉及以下的气敏蛋白:
在原核和真核细胞中结合气体和启动信号级联,
这一类是可溶性鸟苷酸环化酶(sGC)的NO结合血红素结构域,sGC是脑内主要的NO感受器,
人类非天然氨基酸(UAA)是化学合成的分子,
天然存在的氨基酸已被用于帮助研究各种生物学和医学相关的
蛋白质系统UAA具有可化学定制以用于以下应用的明显优势:
感兴趣和位点特异性地掺入蛋白质,同时对蛋白质结构的侵入性最小,
UAA本身并不比天然氨基酸大多少。这个项目利用琥珀密码子
在H-NOX蛋白中掺入非天然氨基酸以研究构象变化的方法
并调节气体结合亲和力。本项目的第一个目标是利用含腈的振动报告子UAA
或叠氮基团来监测两种细菌H-NOX的溶剂化环境和构象变化
proteins.该项目的第二个目的是调节O2结合的氧结合亲和力,
腾冲嗜热厌氧菌H-NOX使用UAA。这项研究采用了一些生物物理
技术(FTIR,停流紫外可见光谱,激光闪光光解,CD光谱,和X射线
晶体学)以分析UAA掺入的H-NOX突变体。这些研究将扩大我们的
了解这些重要的气体结合蛋白,致力于开发一种新的O2输送分子
具有作为治疗剂治疗缺氧相关疾病的潜在用途,
利用非天然氨基酸来研究和/或工程化其他生物医学上的结构和功能,
相关蛋白质
英文摘要
PROJECT SUMMARY.
The heme nitric oxide and/or oxygen binding (H-NOX) proteins form a class of gas sensing proteins involved in
binding gases and initiating signaling cascades in both prokaryotic and eukaryotic cells with a key member of
this class being the NO-binding heme domain of soluble guanylate cyclase (sGC), the main NO sensor in
humans. Unnatural amino acids (UAAs) are chemically synthesized molecules with minor alterations from
naturally occurring amino acids that have been used to help study various biologically and medically relevant
protein systems. UAAs have the distinct advantage of being chemically customizable for an application of
interest and site-specifically incorporated into proteins while being minimally invasive of protein structure as the
UAAs themselves are not much larger than native amino acids. This project utilizes the amber codon
methodology to incorporate unnatural amino acids in H-NOX proteins to both study conformational changes
and tune gas-binding affinity. The first aim of this project is to utilize vibrational reporter UAAs containing nitrile
or azido groups to monitor solvation environments and conformational changes of two bacterial H-NOX
proteins. The second aim of this project is to tune the oxygen-binding affinity of the O2-binding
Thermoanaerobacter tencongensis H-NOX using UAAs. This research employs a number of biophysical
techniques (FTIR, stopped-flow UV-Visible spectroscopy, laser flash photolysis, CD spectroscopy, and X-ray
crystallography) to analyze the UAA-incorporated H-NOX mutants. These studies will extend our
understanding of these important gas-binding proteins, work towards developing a new O2 delivery molecule
with potential use as a therapeutic agent to treat hypoxia-related diseases, and expand the applications and
utility of unnatural amino acids to study and/or engineer the structure and function of other biomedically
relevant proteins.
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