New technology transfer----Nicoya's new generation SPR technology in the study of calmodulin and NOS

September 06, 2023

Study on the interaction between EF chiral pair mutant calmodulin and nitric oxide synthase binding domain peptide

Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

Nitric oxide synthase ( NOS) is a very important biosynthesis catalyst for NO synthesis. Nitric oxide (NOS ) includes three isoforms: neuronal enzyme ( nNOS), and endothelial nitric oxide synthase ( eNOS) and inducible nitric oxide synthase (iNOS). The NO produced by each enzyme is used for neurotransmission, vasodilation, and immune response, respectively. Nitric oxide synthase is a homodimer formed by the N-terminal oxidase domain and the c-terminal binding domain through the calmodulin structure threshold. protein.

Nitric oxide synthase ( NOS) catalyzes the production of NO by L-arginine and plays a major role in several key physiological processes. Calmodulin (CaM) is a small ubiquitinated Ca ⁺² coupled protein. to the activation (NOS); calmodulin binding EF-hand Ca ⁺² by changes, but calmodulin activation mechanism NOS is not entirely clear at present for CaM interaction with NOS method complicated, time-consuming. For this reason, John waterloo University in Canada by LSPR niocya latest development of real-time label-free detection technology to analyze the different chiral mutated CaM interaction with NOS, NOS contrast with CaM binding affinity for CaM- obtained by a simple method The kinetic data of the NOS effect were good, revealing that CaM activates NOS by EF chiral pair mutations ! The results were shared with the research methodology at the 2017 Chemical Biophysics Symposium.

CaM is a small, highly dynamic, protein that catalyzes Ca ²⁺ binding and is essential for NOS activity in almost all eukaryotic organisms . It consists of a spherical N-terminal and c-terminal domain linked by a variable central junction region, each region CaM binding to Ca ²⁺ via a 2 chiral motif , which can bind 4 Ca ²⁺ , these motifs It is necessary for the mutual binding and the activity of NOS .

FIG as: Compact Ca2 + calmodulin binding different state (A) Apo-CaM (B ) HOLO-CaM (C) HOLO-CaM structure

The picture shows the structure of the different CaM EF chiral mutations .

Based on some information obtained from previous experiments, the John team used Nicoya's LSPR patented technology product OpenSPR real-time mark-free technology.

The activation mechanism of CaM and NOS was detected. The CaM, which was verified by electrophoresis, was purified and identified by mass spectrometry, and then used for interaction with NOS.

Figure: Results of SDS-PAGE analysis after CaM purification

Protein binding assay: The surface of the nano-gold chip fixed OpenSPR of NOS, and do CaM concentration gradient detection, analysis results into TraceDrawer.

(Fig. 4) OpenSPR real-time detection , the concentration of wild-type CaM on nNOS (red line) and eNOS peptide (purple red line ) was detected at different concentrations after gradient dilution , and the whole process of binding, dissociation and regeneration was observed . The results showed that the experimental curve was stable. And the signal is significant.

(Fig. 5) The data of CaM and cNOS binding of different mutations were imported into TRACE DRAWER software to obtain Koff, Kon and KD values. The results are as follows:

The results show: CaMcc with higher affinity than cNOS wt CaM, relatively wt CaM, all except nCaM CaM mutant and CaMcc, NOS and dissociation rates are accelerated, and the results obtained with previous conventional methods can activate only CaMc NOS, Other The results of poor or inactive activation are consistent, which also proves the accuracy of OpenSPR in the study of CaM and cNOS binding, and the detection method is simple and the data is rich. The mechanism of CaM activation cNOS activation is due to the occurrence of EF in CaM. Chiral pair mutations, using this method to continue to verify the mechanism of CaM activation of eNOS and iNOS .

(1) Alderton et al. Biochem. J. (2001) 357:593

(2) Babu YS, Bugg CE, Cook WJ: J. Mol. Bio. (1988) 204: 191-204

(3) Spratt.DEet FEBS (2006) 273, 1759-1771

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