A chemometric study combined with spectroscopy for the quantification of secondary structure of flagellar‐associated protein 174 (FAP174)

Abstract

The secondary structure analyses of proteins hold immense importance in the field of protein science because it plays a vital role in its hierarchical classification. It is the most important transitional step in the prediction of the threedimensional structure of any protein. The aim of the current study is to quantitatively determine the secondary structure of an important ciliary protein, viz, an MYC-binding protein-1 (MYCBP-1) orthologue, viz, flagellar-associated protein 174 (FAP174) from the green alga, Chlamydomonas reinhardtii. FAP174 binds to A-kinase anchoring protein 240 (AKAP240) and has a crucial role to play in ciliary motility. Hence, the biochemical characterization of FAP174 becomes more vital in understanding its molecular function. The accurate secondary structure of FAP174 was investigated through circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) combined with partial least square regression (PLSR) methods. The far-UV CD spectrum of FAP174 exhibited a positive band at 192 nm and negative bands at 208 and 221 nm, whereas quantification through BeStSel web server revealed 65% α-helix, approximately 2% of antiparallel β-sheets, 6% of β-turns, and 27% of unordered structures. These results were further confirmed by FTIR spectrum of FAP174 that revealed amide I and II bands at 1654 and 1547 cm −1 , respectively. And the PLSR calibration models led to the quantification of the secondary structure for FAP174 protein that fairly corroborated with the values obtained by quantifying CD spectrum, these being approximately 54% α-helix, 0% β-sheets, approximately 12% β-turns, and approximately 34% other structures. The recombinant FAP174 protein is therefore considerably α-helical and has negligible or no β-sheets. K E Y W O R D Schemometric methods, circular dichroism, flagellar associated protein 174, Fourier transform infrared spectroscopy, partial least square regression