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。
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.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
海外基金