Structural and biophysical properties of h-FANCI ARM repeat protein.
Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Mohd Quadir
MQ
Siddiqui
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Rajan Kumar
RK
Choudhary
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Pankaj
P
Thapa
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Neha
N
Kulkarni
b Molecular Biology Division , Bhabha Atomic Research Centre , Mumbai 400 085 , India.
Yogendra S
YS
Rajpurohit
b Molecular Biology Division , Bhabha Atomic Research Centre , Mumbai 400 085 , India.
Hari S
HS
Misra
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Nikhil
N
Gadewal
c Department of Biochemistry & Bioinformatics Centre , Mahatma Gandhi Institute of Medical Sciences , Sevagram ( Wardha ) 442102 , India.
Satish
S
Kumar
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Syed K
SK
Hasan
a Tata Memorial Centre, Advanced Centre for Treatment , Research and Education in Cancer , Kharghar, Navi Mumbai , Maharashtra 410 210 , India.
Ashok K
AK
Varma
D002942:Circular Dichroism; D051856:Fanconi Anemia Complementation Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
C519921:FANCI protein, human; D051856:Fanconi Anemia Complementation Group Proteins; D011994:Recombinant Proteins
ARM repeats; helix-turn-helix structural motif; human-FANCI