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Experimental and Computational Solid-State NMR Methods for Structure Determination of the Membrane Protein EmrE
Experimental and Computational Solid-State NMR Methods for Structure Determination of the Membrane Protein EmrE
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20260202103635
- ISBN
- 9798286456499
- DDC
- 574.191
- 서명/저자
- Experimental and Computational Solid-State NMR Methods for Structure Determination of the Membrane Protein EmrE
- 발행사항
- [Sl] : The University of Wisconsin - Madison, 2025
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2025
- 형태사항
- 334 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 87-01, Section: B.
- 주기사항
- Advisor: Rienstra, Chad M.
- 학위논문주기
- Thesis (Ph.D.)--The University of Wisconsin - Madison, 2025.
- 초록/해제
- 요약Nuclear magnetic resonance (NMR) spectroscopy is a powerful biophysical technique to probe the structure and dynamics of biomolecules at an atomic level. Solid-state NMR (SSNMR) is particularly effective at characterizing structural and dynamic properties of membrane proteins which are challenging to study by other structural biology techniques. Membrane protein structure determination involves several steps starting with data collection and processing, resonance assignments, and structure calculations. This thesis focuses on SSNMR method development to with application to calculate a high-resolution structure of the membrane protein EmrE implicated in antibiotic resistance. Chapters 2 and 3 focus on maximizing and enhancing spectrometer stability. First, an experimental method is introduced to minimize temperature dependence of amplifiers and maximize cross-polarization stability, enabling 3D spectra of EmrE to be collected over several days. Next, principal component analysis (PCA) is employed to monitor the stability of spectrometers and improve the sensitivity of multidimensional spectra. Backbone assignments of EmrE were then assigned using solution and SSNMR and sidechains of EmrE were assigned using SSNMR (chapter 4). Resonance assignments allowed for distance restraints to be obtained to drive a high-resolution structure (0.8 A) calculation of EmrE. 13C13C distance restraints were obtained from Xplor-NIH's program probabilistic assignment for structure determination (PASD), 13C15N restraints from transferred-echo double resonance (TEDOR) experiments, and 13C water accessibility from 2D 13C13C T2 filtered experiments. Finally, we introduce a new structure validation approach in which a protein structural model is used to simulate multidimensional NMR spectra and scored against experimental spectra.
- 일반주제명
- Biophysics
- 일반주제명
- Analytical chemistry
- 일반주제명
- Biochemistry
- 일반주제명
- Bioengineering
- 키워드
- Proteins
- 키워드
- Solid-state NMR
- 기타저자
- The University of Wisconsin - Madison Biochemistry-ALS
- 기본자료저록
- Dissertations Abstracts International. 87-01B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520260202103635
■006m o d
■007cr#unu||||||||
■020 ▼a9798286456499
■035 ▼a(MiAaPQ)AAI32047582
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574.191
■1001 ▼aHarding, Benjamin D.
■24510▼aExperimental and Computational Solid-State NMR Methods for Structure Determination of the Membrane Protein EmrE
■260 ▼a[Sl]▼bThe University of Wisconsin - Madison▼c2025
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2025
■300 ▼a334 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 87-01, Section: B.
■500 ▼aAdvisor: Rienstra, Chad M.
■5021 ▼aThesis (Ph.D.)--The University of Wisconsin - Madison, 2025.
■520 ▼aNuclear magnetic resonance (NMR) spectroscopy is a powerful biophysical technique to probe the structure and dynamics of biomolecules at an atomic level. Solid-state NMR (SSNMR) is particularly effective at characterizing structural and dynamic properties of membrane proteins which are challenging to study by other structural biology techniques. Membrane protein structure determination involves several steps starting with data collection and processing, resonance assignments, and structure calculations. This thesis focuses on SSNMR method development to with application to calculate a high-resolution structure of the membrane protein EmrE implicated in antibiotic resistance. Chapters 2 and 3 focus on maximizing and enhancing spectrometer stability. First, an experimental method is introduced to minimize temperature dependence of amplifiers and maximize cross-polarization stability, enabling 3D spectra of EmrE to be collected over several days. Next, principal component analysis (PCA) is employed to monitor the stability of spectrometers and improve the sensitivity of multidimensional spectra. Backbone assignments of EmrE were then assigned using solution and SSNMR and sidechains of EmrE were assigned using SSNMR (chapter 4). Resonance assignments allowed for distance restraints to be obtained to drive a high-resolution structure (0.8 A) calculation of EmrE. 13C13C distance restraints were obtained from Xplor-NIH's program probabilistic assignment for structure determination (PASD), 13C15N restraints from transferred-echo double resonance (TEDOR) experiments, and 13C water accessibility from 2D 13C13C T2 filtered experiments. Finally, we introduce a new structure validation approach in which a protein structural model is used to simulate multidimensional NMR spectra and scored against experimental spectra.
■590 ▼aSchool code: 0262.
■650 4▼aBiophysics
■650 4▼aAnalytical chemistry
■650 4▼aBiochemistry
■650 4▼aBioengineering
■653 ▼aMagic angle spinning
■653 ▼aNuclear magnetic resonance
■653 ▼aProteins
■653 ▼aStructural biology
■653 ▼aSolid-state NMR
■690 ▼a0786
■690 ▼a0202
■690 ▼a0486
■690 ▼a0487
■71020▼aThe University of Wisconsin - Madison▼bBiochemistry-ALS.
■7730 ▼tDissertations Abstracts International▼g87-01B.
■790 ▼a0262
■791 ▼aPh.D.
■792 ▼a2025
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17358044▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
