Structural features of FAP174, a MYCBP-1 orthologue from Chlamydomonas reinhardtii, revealed by computational and experimental analyses

Yogesha, M; Rao, Venkatramanan G.; Martis, Elvis A. F.; Coutinho, Evans C.; Gohlke, Holger; Chidangil, Santhosh; Dongre, Prabhakar M.; D’Souza, Jacinta S.

doi:10.1039/c7ra07836f

Cited by 2 publications

(13 citation statements)

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“…The protein formed dimers, and both the monomer and dimer species stay in equilibrium post the purification step. 57 In order to solve both secondary and tertiary structures of this protein, several attempts of proton-NMR using varying buffer conditions with the recombinant FAP174 protein yielded in poor spectra (data not shown). Parallel attempts to crystallize FAP174 have also failed, and the current study primes exclusively towards getting a better strategy of quantifying the secondary structure of a protein that has proven difficulty in structural characterization.…”

Section: Resultsmentioning

confidence: 99%

“…The molecular size of the pure protein was ascertained using MALDI-TOF analysis in a recent report. 57…”

Section: Purification Of Fap174 Proteinmentioning

confidence: 99%

“…The resultant spectral values are expressed in units of millidegrees (mdeg). 57 The secondary structure of FAP174 protein was analyzed by deconvolution of the CD spectrum using the online BeStSel analysis program 10 applied at the wavelength range of 190 to 250 nm.…”

Section: Spectroscopymentioning

confidence: 99%

“…This confirmed the α-helical nature of the recombinant FAP174 protein. 57 Upon deconvolution of the recombinant FAP174 protein spectrum, its secondary structure could be quantified using BeStSel web server. This web server provides a reference set that contains CD spectra of 73 proteins and is efficient in calculating eight different secondary structures, namely, regular helix-middle part of the helices (H1), distorted helix-2-2 residues at the end of the α-helix (H2), antiparallel β-sheets (left twisted, relaxed, and right twisted), parallel β-sheets, turns, and other structures (3 10 helix, π-helix, β-bridge, bend, and loop of the structure).…”

Section: Quantification Of Fap174 Secondary Structure Content Usingmentioning

confidence: 99%

“…Predicted tertiary structure of FAP174 WT comprises five helices (A-E); it has a dimerization and docking domain and an oligomerization control motif present in helices B and C, respectively. 57 To the best of our knowledge, no study has been carried out on the structural determination of FAP174 using PLS regression method. The current study focuses on quantifying the percentage of secondary structure of the purified recombinant FAP174 protein using two different approaches.…”

Section: Introductionmentioning

confidence: 99%

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

Yogesha

Rao

Devangad

et al. 2020

Journal of Chemometrics

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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

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Section: Resultsmentioning

confidence: 99%

“…The molecular size of the pure protein was ascertained using MALDI-TOF analysis in a recent report. 57…”

Section: Purification Of Fap174 Proteinmentioning

confidence: 99%

Section: Spectroscopymentioning

confidence: 99%

Section: Quantification Of Fap174 Secondary Structure Content Usingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Yogesha

Rao

Devangad

et al. 2020

Journal of Chemometrics

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A‐kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii

Rao,

Shendge,

D'Gama

et al. 2024

FEBS Letters

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Cilia are microtubule‐based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A‐kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central‐pair microtubules of Chlamydomonas reinhardtii cilia uses two C‐terminal amphipathic helices to bind to its partner FAP174, an RIIα‐like protein with a D/D domain at the N‐terminus. Co‐immunoprecipitation using anti‐FAP174 antibody with an enriched central‐pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub‐ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central‐pair microtubules.

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Structural features of FAP174, a MYCBP-1 orthologue from Chlamydomonas reinhardtii, revealed by computational and experimental analyses

Abstract: The ciliary MYCBP-1 (FAP174) from Chlamydomonas reinhardtii is an R2D2 protein and harbors a Dimerization and Docking domain.

Cited by 2 publications

References 52 publications

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

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

A‐kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii

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