STRUCTURAL AND FUNCTIONAL ANALYSIS OF DISEASE CAUSING MUTATIONS IN THE M10 DOMAIN OF TITIN

Titin (3-4MDa) is the largest monomeric protein found in humans and is crucial for the overall structural stability and function of muscle tissues. Dubbed the “molecular ruler”, titin sets the length of the sarcomere while ensuring the structure is organized. While titin binds to many targets, the extreme C-terminal domain (M10) plays an important role by binding to the extreme N-terminal domain (Ig1) of the giant muscle protein obscurin. Mutations in the M10 domain of titin have been linked to cases of limb girdle muscular dystrophy 2J (LGMD2J), most likely due to an ablation of M10-Ig1 binding. Here, we have recombinantly expressed and purified wild type M10 and all known human M10 mutations. Initial structural characterization using Circular Dichroism (CD) indicates that while the wild-type domain is predominantly beta sheet, most mutations are only partially folded, although each has a distinct structural phenotype. Isothermal Titration Calorimetry (ITC) studies demonstrate that misfolded mutants do not bind to Ig1, however one mutant (Belgian) binds to Ig1 with a similar affinity to wild-type. Protein NMR spectroscopy confirms these findings, while revealing that the Belgian mutation exists in two conformations, one closely resembling wild type, and one showing distinct structural differences. Currently, both wild-type and mutant NMR spectra are being sequence-specifically assigned, allowing for the identification of the distinct chemical differences between wild-type and the two conformations of the Belgian mutation.

Additional Abstract Information

Student(s): Michael W. Rudloff

Department: Chemistry and Biochemistry

Faculty Advisor: Dr. Michael W. Rudloff

Type: Oral

Year: 2014