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Chemistry

의 NMR 공명 확인하는 방법 Published: December 12, 2013 doi: 10.3791/50875
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1, 1, 1, 1
1Department of Chemistry, Louisiana State University

Abstract

핵 자기 공명 (NMR) 분광법은 단백질 구조 및 동적 연구 입증 된 기술이다. NMR로 단백질을 연구하기 위해, 안정적인 자기 동위 원소는 일반적으로 감도를 개선하고 순차적 공명 할당을 허용 대사 포함된다. 환원 13 C-메틸화를 통해 표현 박테리아 숙주, 동위 원소 결합하는 가장 일반적인 방법에 순종하지 않는 단백질에 대한 대체 표시 방법입니다. 환원성 13 C-메틸화는 13 C-디메틸 아미노 그룹으로의 단백질의 일차 아미노기 (라이신의 ε-아미노기와 N-말단 α-아미노기를) 수정 온화한 조건 하에서 수행 화학 반응이다. 대부분의 단백질의 구조와 기능은 그 대사 라벨링에 대한 실행 가능한 대안 만들기, 수정에 의해 변경되지 않습니다. 환원 13 C-메틸화는 스파 스, 동위 원소 라벨, N을 할당하는 전통적인 방법을 추가하기 때문에MR 신호는 적용되지 않습니다. 단백질 (13) C-디메틸 NMR 신호의 할당을 돕기 위해 질량 분석기 (MS)를 사용하여 대안의 할당 방법이 개발되었다. 이 방법은 각각의 차 아미노기에 13 C-라벨의 부분과 다른 양에 의존한다. Lys1의 α-및 ε-다이 메틸 아미노 그룹이 개별적으로 MS로 측정 할 수 없기 때문에, 단백질의 N-말단 잔기가 리신 때 방법의 제한이 발생한다. 이 한계를 회피하기 위해,이 방법은 N-말단 α-아민 및 측쇄 ε-아민 모두와 연관된 13 C-NMR dimethylamines의 공진을 식별하는 기술된다. N-말단 α-디메틸 아민과 암탉 흰자 리소자임, Lys1의 사이드 체인 ε-디메틸 아민의 NMR 신호는 1 H-13 C 헤테로 핵 단일 양자 간섭 스펙트럼에서 확인된다.

Materials

Name Company Catalog Number Comments
Acetonitrile Sigma 34851
Amicon Ultra 4 ml centrifugal filter (3k MWCO) Fisher Scientific UFC800308 (8pk)
Aminopeptidase Creative Biomart Aminopeptidase-86A
Ammonium bicarbonate Sigma A6141
Ammonium sulfate Sigma A5132
Borane-dimethylamine complex Sigma 180238 Make fresh 1 M solution before use.
Boric acid Sigma B6768
Bovine pancreas insulin Sigma I5500
C18 Spin Columns Thermo Scientific 89870
Deuterium oxide Cambridge Isotope Laboratories DLM-4-100
1,2-dichloroethane-13C2 Sigma 714321 80 mM stock solution stored at -80 °C. Used as reference in NMR spectra at 24 mM.
Formaldehyde (36.5% wt/wt) Sigma 33220 Make fresh 1 M solution before use.
13C-formaldehyde (99%) Cambridge Isotope Laboratories CLM-806-1 Make fresh 1 M solution before use.
Lysozyme (hen egg white) Sigma L6376
Sodium phosphate (dibasic heptahydrate) Sigma S9390
Sodium phosphate (monobasic) Sigma S9638
Tricine Sigma T0377
Trifluoroacetic acid Sigma 302031
700 MHz Varian VNMRS with 5 mm HCN 5922 probe Agilent Technologies
Bruker UltrafleXtreme MALDI TOF/TOF Bruker

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의 NMR 공명 확인하는 방법<sup&gt; 13</sup환원 메틸을 단백질에&gt; C-디메틸 N-말단 아민
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Cite this Article

Roberson, K. J., Brady, P. N.,… More

Roberson, K. J., Brady, P. N., Sweeney, M. M., Macnaughtan, M. A. Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins. J. Vis. Exp. (82), e50875, doi:10.3791/50875 (2013).

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