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
项目总结。
血红素一氧化氮和/或氧结合蛋白(H-NOX)形成一类气体敏感蛋白,参与
结合气体和启动信号级联在原核和真核细胞与关键成员
这个类是可溶性鸟苷环化酶(SGC)的NO结合的血红素结构域,是人体内主要的NO感受器。
人类。非天然氨基酸(UAA)是化学合成的分子,只需稍作改动即可
天然氨基酸已被用来帮助研究各种生物和医学上的相关
蛋白质系统。UAA具有独特的优势,即可针对以下应用进行化学定制
兴趣和位点特异性地结合到蛋白质中,同时对蛋白质结构的侵袭最小,因为
尿酸本身并不比天然氨基酸大多少。这个项目使用了琥珀密码子
在H-NOX蛋白中加入非天然氨基酸研究构象变化的方法学
并调整气体结合亲和力。这个项目的第一个目标是利用含有丁腈的振动报告UaAs
或叠氮基团监测两个细菌H-NOX的溶剂化环境和构象变化
蛋白质。该项目的第二个目标是调节O2-结合的氧结合亲和力
利用UAAS产热厌氧菌产H-NOX。这项研究使用了许多生物物理学
技术(FTIR、停流紫外可见光谱、激光闪光光解、CD光谱和X射线
结晶学)分析掺入UAA的H-NOX突变体。这些研究将延长我们的
了解这些重要的气体结合蛋白,致力于开发一种新的氧气输送分子
在治疗缺氧相关疾病方面具有潜在的用途,并扩大了其应用和
非天然氨基酸在研究和/或设计其他生物医学结构和功能方面的应用
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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