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

• Published in: Journal of biological inorganic chemistry 2024-03, Vol.29 (2), p.243-250
• Main Authors: Singh, Swapnil, Gyawali, Yadav Prasad, Jiang, Ting, Bukowski, Gregory S., Zheng, Huayu, Zhang, Haikun, Owopetu, Rebecca, Thielges, Megan C., Feng, Changjian
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Calmodulin; Calmodulin - chemistry; Calmodulin - metabolism; Electron transfer; Heme; Infrared spectroscopy; Life Sciences; Microbiology; Molecular Docking Simulation; Nitric oxide; Nitric Oxide Synthase Type II - chemistry; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide synthase; Original Paper; Oxygenase; Phenylalanine; Protein Binding; Proteins; Spectrophotometry, Infrared; Spectrum analysis
• ISSN: 1432-1327; 0949-8257; 1432-1327
• DOI: 10.1007/s00775-024-02046-0

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 ( CN F) 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 CN F at residue 108, which is at the putative CaM–heme (NOS) docking interface. CN F 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. Graphical abstract