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      Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

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          Abstract

          In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C− 1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T 2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.

          Supplementary Information

          The online version contains supplementary material available at 10.1007/s10858-023-00427-2.

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          NMRPipe: a multidimensional spectral processing system based on UNIX pipes.

          Frank Delaglio, Stephan Grzesiek, GeertenW. Vuister … (1995)
          The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
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            The CCPN data model for NMR spectroscopy: development of a software pipeline.

            Wim Vranken, Wayne Boucher, Tim J. Stevens … (2005)
            To address data management and data exchange problems in the nuclear magnetic resonance (NMR) community, the Collaborative Computing Project for the NMR community (CCPN) created a "Data Model" that describes all the different types of information needed in an NMR structural study, from molecular structure and NMR parameters to coordinates. This paper describes the development of a set of software applications that use the Data Model and its associated libraries, thus validating the approach. These applications are freely available and provide a pipeline for high-throughput analysis of NMR data. Three programs work directly with the Data Model: CcpNmr Analysis, an entirely new analysis and interactive display program, the CcpNmr FormatConverter, which allows transfer of data from programs commonly used in NMR to and from the Data Model, and the CLOUDS software for automated structure calculation and assignment (Carnegie Mellon University), which was rewritten to interact directly with the Data Model. The ARIA 2.0 software for structure calculation (Institut Pasteur) and the QUEEN program for validation of restraints (University of Nijmegen) were extended to provide conversion of their data to the Data Model. During these developments the Data Model has been thoroughly tested and used, demonstrating that applications can successfully exchange data via the Data Model. The software architecture developed by CCPN is now ready for new developments, such as integration with additional software applications and extensions of the Data Model into other areas of research.
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              Using chemical shift perturbation to characterise ligand binding.

              Mike P. Williamson (2013)
              Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by simultaneous fitting of many measured shift changes, or more simply by adding substoichiometric amounts of ligand. The chemical shift changes can be used as restraints for docking ligand onto protein. By use of quantitative calculations of ligand-induced chemical shift changes, it is becoming possible to determine not just the position but also the orientation of ligands. Copyright © 2013 Elsevier B.V. All rights reserved.
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                Author and article information

                Contributors
                Roman Lichtenecker: roman.lichtenecker@univie.ac.at
                Robert Konrat: robert.konrat@univie.ac.at
                Journal
                Journal ID (nlm-ta): J Biomol NMR
                Journal ID (iso-abbrev): J Biomol NMR
                Title: Journal of Biomolecular Nmr
                Publisher: Springer Netherlands (Dordrecht )
                ISSN (Print): 0925-2738
                ISSN (Electronic): 1573-5001
                Publication date (Electronic): 11 October 2023
                Publication date PMC-release: 11 October 2023
                Publication date (Print): 2024
                Volume: 78
                Issue: 1
                Pages: 1-8
                Affiliations
                [1 ]GRID grid.10420.37, ISNI 0000 0001 2286 1424, Christian Doppler Laboratory for High-Content Structural Biology and Biotechnology, Department of Structural and Computational Biology, Max Perutz Laboratories, , University of Vienna, ; Campus Vienna Biocenter 5, 1030 Vienna, Austria
                [2 ]Christian Doppler Laboratory for High-Content Structural Biology and Biotechnology, Institute of Organic Chemistry, University of Vienna, ( https://ror.org/03prydq77) Währingerstraße 38, 1090 Vienna, Austria
                [3 ]Vienna Doctoral School in Chemistry (DoSChem), University of Vienna, ( https://ror.org/03prydq77) Währingerstraße 42, 1090 Vienna, Austria
                [4 ]GRID grid.10420.37, ISNI 0000 0001 2286 1424, Department of Structural and Computational Biology, , Max Perutz Laboratories, University of Vienna, ; Campus Vienna Biocenter 5, 1030 Vienna, Austria
                [5 ]GRID grid.486422.e, ISNI 0000000405446183, Boehringer Ingelheim RCV GmbH & Co. KG, ; Dr. Boehringer Gasse 5-11, 1121 Vienna, Austria
                Article
                Publisher ID: 427
                DOI: 10.1007/s10858-023-00427-2
                PMC ID: 10981609
                PubMed ID: 37816933
                SO-VID: 36a26b23-1c0e-40bb-b9c8-de443bb2fcaa
                Copyright © © The Author(s) 2023
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                Date received : 5 July 2023
                Date accepted : 28 September 2023
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100006012, Christian Doppler Forschungsgesellschaft;
                Funded by: University of Vienna
                Open Access :

                Open access funding provided by University of Vienna.

                Categories
                Subject: Article
                Custom metadata
                issue-copyright-statement © Springer Nature B.V. 2024

                ScienceOpen disciplines: Molecular biology
                Keywords: precursor labeling,transversal relaxation studies,ch-π interaction,ligand binding
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: precursor labeling, transversal relaxation studies, ch-π interaction, ligand binding

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