title: Human microtubule affinity-regulating kinase 4 is stable at extremes of pH.
abstract: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Farha
authors.initials: F
authors.lastname: Naz
authors.affiliation: b School of Chemistry and Physics , University of Kwa-Zulu Natal , Chiltern Hill, Durban 4000 , South Africa.
authors.forename: Parvesh
authors.initials: P
authors.lastname: Singh
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Md
authors.initials: M
authors.lastname: Imtaiyaz Hassan
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D021122:Protein Subunits; D013057:Spectrum Analysis
chemical_list: D021122:Protein Subunits; C483047:MARK4 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: effect of pH; kinase domain; microtubule affinity-regulating kinase 4; microtubule dynamics; molecular dynamics simulation; protein aggregation