Institute of Crystallography - CNR

Structural characterization of extracellular matrix proteins

Structural characterization of extracellular matrix proteins (amino-terminal domain of alpha dystroglycan, beta dystroglycan and LG1-2 domains of agrin) and their pathological mutants.

Cloning, mutagenesis, recombinant expression of the LG1-2 domains of human agrin in a soluble form in fusion with Trx, purification by chromatographic systems, proteolytic removal of the tag and purification. Purification by chromatographic systems of the amino-terminal domain of murine alpha dystroglycan and beta dystroglycan and their pathological mutants Biophysical and structural characterization of the effect on the conformation of these extracellular matrix proteins of pathological mutations by molecular dynamics, limited proteolysis, DSF, Fluorescence, X-ray crystallography and SAXS.

Reference works

– Signorino G., Covaceuszach S., Bozzi M., Hübner W., Mönkemöller V., Konarev P.V., Cassetta A., Brancaccio A., Sciandra F. A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein. Hum Mutat. (2018) Feb;39(2):266-280.
– Covaceuszach S., Bozzi M., Bigotti M.G., Sciandra F., Konarev P.V., Brancaccio A., Cassetta A. “The effect of the pathological V72I, D109N and T190M missense mutations on the molecular structure of α-dystroglycan.” PLoS One. (2017) Oct 16;12(10):e0186110. doi: 10.1371/journal.pone.0186110. eCollection 2017.
– Covaceuszach S., Bozzi M., Bigotti M.G. , Sciandra F., Konarev P., Brancaccio A., Cassetta A. Structural flexibility of human alpha-Dystroglycan. FEBS Open bio 7:1064 (2017), DOI: 10.1002/2211-5463.12259.
– Bozzi M., Cassetta A., Covaceuszach S., Bigotti M.G. , Bannister S., Hübner W., Sciandra F.,Lamba D., Brancaccio A. “The Structure of the T190M Mutant of Murine alpha-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy.” PLoS One (2015) 10(5):e0124277

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