Solid-state NMR spectroscopy (ssNMR) is uniquely suited for atomic-resolution structural investigations of large protein assemblies, which are notoriously difficult to study due to their insoluble and non-crystalline nature. However, assignment ambiguities because of limited resolution and spectral crowding are currently major hurdles that quickly increase with the length of the polypeptide chain. The line widths of ssNMR signals are independent of proteins size, making segmental isotope labeling a powerful approach to overcome this limitation. It allows a scalable reduction of signal overlap, aids the assignment of repetitive amino acid sequences, and can be easily combined with other selective isotope labeling strategies. Here we present a detailed protocol for segmental isotope labeling of insoluble proteins using protein trans-splicing. Our protocol exploits the ability of many insoluble proteins, such as amyloid fibrils, to fold correctly under in vitro conditions. In combination with the robust trans-splicing efficiency of the intein DnaE from Nostoc punctiforme, this allows for high yields of segmentally labeled protein required for ssNMR analysis.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-4939-6451-2_10 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Catalyst-Free Nitrogen Fixation by Microdroplets through a Radical-Mediated Disproportionation Mechanism under Ambient Conditions.
J Am Chem Soc
January 2025
State Key Laboratory of Physical Chemistry of Solid Surfaces, iChEM, College of Chemistry and Chemical Engineering, Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province (IKKEM), Xiamen University, Xiamen 361005, China.
Nitrogen fixation is essential for the sustainable development of both human society and the environment. Due to the chemical inertness of the N≡N bond, the traditional Haber-Bosch process operates under extreme conditions, making nitrogen fixation under ambient conditions highly desirable but challenging. In this study, we present an ultrasonic atomizing microdroplet method that achieves nitrogen fixation using water and air under ambient conditions in a rationally designed sealed device, without the need for any catalyst.
View Article and Find Full Text PDFNovel Chlorin with a HYNIC: Synthesis, Tc-Radiolabeling, and Initial Preclinical Evaluation.
Molecules
December 2024
M. V. Lomonosov Institute of Fine Chemical Technology, MIREA-Russian Technological University, 86 Vernadsky Av., 119571 Moscow, Russia.
The use of radiopharmaceuticals for diagnostics in oncology allows for the detection of the disease at an early stage. Among diagnostic radionuclides, Tc is a promising isotope that has been used to create several drugs for clinical use. One of the most effective Tc chelators is 6-hydrazinylnicotinic acid (HYNIC), which, when combined with various vector molecules, can be used for targeted delivery of radionuclides to tumor tissues.
View Article and Find Full Text PDFFailure to repair damaged NAD(P)H blocks de novo serine synthesis in human cells.
Cell Mol Biol Lett
January 2025
Enzymology and Metabolism Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367, Belvaux, Luxembourg.
Background: Metabolism is error prone. For instance, the reduced forms of the central metabolic cofactors nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), can be converted into redox-inactive products, NADHX and NADPHX, through enzymatically catalyzed or spontaneous hydration. The metabolite repair enzymes NAXD and NAXE convert these damaged compounds back to the functional NAD(P)H cofactors.
View Article and Find Full Text PDFUltrasensitive Characterization of Native Bacterial Biofilms via Dynamic Nuclear Polarization-Enhanced Solid-State NMR.
Angew Chem Int Ed Engl
January 2025
University of Pittsburgh School of Medicine, Structural Biology, 3501 5th Ave., Biomedical Science Tower 3, Room 2044, 15261, Pittsburgh, UNITED STATES OF AMERICA.
Bacterial biofilms are major contributors to persistent infections and antimicrobial resistance, posing significant challenges to treatment. However, obtaining high-resolution structural information on native bacterial biofilms has remained elusive due to the methodological limitations associated with analyzing complex biological samples. Solid-state NMR (ssNMR) has shown promise in this regard, but its conventional application is hindered by sensitivity constraints for unlabeled native samples .
View Article and Find Full Text PDFDetection of amino thiols from fish samples by stable isotope chemical labelling coupled with ultra-performance liquid chromatography-tandem mass spectrometry.
Food Chem
December 2024
Key Laboratory of Aquatic product, Ministry of Agriculture and Rural affairs, National R&D Center for Aquatic Product Processing, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510330, China; Key Laboratory of Efficient Utilization and Processing of Marine Fishery Resources of Hainan Province, Sanya Tropical Fisheries Research Institute, Sanya 572426, China.
The amino thiols are key antioxidants in organisms, and their detection in food is of significant importance. This study developed a new stable isotope chemical labelling coupled with ultra-performance liquid chromatography-tandem mass spectrometry method to detect six amino thiols from fish samples. By the proposed method, amino thiols were labeled after liquid extraction using the stable isotope labeling reagents of iodoacetamide (IAM) and D-IAM.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!