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Probing calmodulin–NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation

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Abstract

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM–NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-l-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM–heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM–NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

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Abbreviations

NOS:

Nitric oxide synthase

iNOS:

Inducible NOS

nNOS:

Neuronal NOS

eNOS:

Endothelial NOS

CaM:

Calmodulin

oxyFMN:

Bidomain oxygenase/FMN construct in which only the oxygenase and FMN domains are present, along with the CaM binding linker region

CNF108 CaM:

CNF is introduced at 108 site on helix E within the C-lobe

CNF29 CaM:

CNF is introduced at 29 site on helix B within the N-lobe

p-iNOS:

Peptide containing the CaM binding region of iNOS

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Acknowledgements

We thank Dr. Henning Tidow for sharing the pET28aCaMC3His plasmid. Computational aspects of this research were supported in part by Lilly Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute.

Funding

This material is based upon work funded by NIH GM133973 and NSF 2041692 (C.F.) and NIH GM148290 and DESC0018983 (M.C.T.). This work was also supported in part by NIH P20GM130422 and P30ES032755.

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Author notes

  1. Swapnil Singh and Yadav Prasad Gyawali contributed equally.

Authors and Affiliations

  1. Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA

    Swapnil Singh, Gregory S. Bukowski & Megan C. Thielges

  2. Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA

    Yadav Prasad Gyawali, Ting Jiang, Huayu Zheng, Haikun Zhang, Rebecca Owopetu & Changjian Feng

  3. Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM, 87131, USA

    Rebecca Owopetu & Changjian Feng

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  1. Swapnil Singh
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Accession Codes: Human iNOS, P35228.

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775_2024_2046_MOESM1_ESM.pdf

Protein expression and purification, LC−MS/MS analysis, NOS enzymatic activity assay, mass spectroscopy, IR sample preparation, FT IR measurements and analysis, MD simulations details and analysis (Figures S1−S19 and Tables S1−S7) (PDF 3515 KB)

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Singh, S., Gyawali, Y.P., Jiang, T. et al. Probing calmodulin–NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation. J Biol Inorg Chem (2024). https://doi.org/10.1007/s00775-024-02046-0

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