An Investigation of the Neuronal NOS and Calmodulin Interactions Following Cross-linking and Proteolysis Using MALDI-MS
Zamler, Julia L.
MetadataShow full item record
As a result of the varied physiological and pathological involvement of -NO, regulation of Nitric oxide synthase (NOS) has been at the forefront of many investigations. The three isoforms of this enzyme belong to a group of calmodulin (CaM) binding proteins that require calcium-bound CaM for activation. Calmodulin has been shown to be the trigger that allows for electron transfer between the two domains of the NOS molecule. For this project, the neuronal isoform of NOS (nNOS) was used. The objective of this study was to investigate if information regarding the site of interaction and orientation of CaM to nNOS binding could be ascertained following cross-linking of the two proteins, their digestion using various proteases, and analysis by mass spectrometry. Cross-linking of the CaM to nNOS was done using formaldehyde. The digestion was done using trypsin, chymotrypsin and V8 protease and mass spectrometry was done using a MALDI-TOF instrument. The results from these studies showed that the combination of cross-linking with formaldehyde and digestion with trypsin will not give accurate data because the lysine amino acids of nNOS were modified during crosslinking. Trypsin cuts at basic residues (lysine and arginine), therefore, if the lysines had been modified, the peptide cutting data would not be accurate.