Huntingtin interacting protein HYPK is intrinsically unstructured.
To characterize HYPK, originally identified as a novel huntingtin (Htt) interacting partner by yeast two hybrid assay, we used various biophysical and biochemical techniques. The molecular weight of the protein, determined by gel electrophoresis, was found to be about 1.3-folds ( approximately 22 kDa) higher than that obtained from mass spectrometric analysis (16.9 kDa). In size exclusion chromatography experiment, HYPK was eluted in three fractions, the hydrodynamic radii for which were calculated to be approximately 1.5-folds (23.06 A) higher than that expected for globular proteins of equivalent mass (17.3 A). The protein exhibited predominantly (63%) random coil characteristics in circular dichroism spectroscopy and was highly sensitive to limited proteolysis by trypsin and papain, indicating absence of any specific domain. Experimental evidences with theoretical analyses of amino acids composition of HYPK and comparison with available published data predicts that HYPK is an intrinsically unstructured protein (IUP) with premolten globule like conformation. In presence of increasing concentration of Ca(2+), HYPK showed conformational alterations as well as concomitant reduction of hydrodynamic radius. Even though any link between the natively unfolded nature of HYPK, its conformational sensitivity towards Ca(2+) and interaction with Htt is yet to be established, its possible involvement in Huntington's disease pathogenesis is discussed.
Raychaudhuri
Swasti
S
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.
Majumder
Pritha
P
Sarkar
Somosree
S
Giri
Kalyan
K
Mukhopadhyay
Debashis
D
Bhattacharyya
Nitai P
NP
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000209:etiology* / Q000235:genetics / Q000473:pathology; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010206:Papain / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D003432:Cross-Linking Reagents; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D014357:Trypsin; D010206:Papain; D002118:Calcium