Abstract
The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single-molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I-band of a sarcomere is composed of about 40 Ig-domains and is exposed to force under normal physiological conditions and connects the free-hanging ends of the myosin filaments to the Z-disc. Recent single-molecule force spectroscopy data show a mechanical hierarchy in the I-band domains. Domains near the C-terminus in this region unfold at forces two to three times greater than domains near the beginning of the I-band. Though all of these Ig-domains are thought to share a fold and topology common to members of the Ig-like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I-band Ig domain to specific, conserved physical-chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into "strong" and "weak" families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the beta-sandwich fold, and force sensitive residues are not only confined to the A'-G region.