Mechanical forces play a fundamental role in protein function, influencing how proteins fold, unfold (or stretch), and interact in biological systems. Specific sequence motifs and structural folds have evolved to sense or bear these forces, enabling proteins to perform their mechanical roles in processes such as adhesion and muscle contraction. Although β-sheet proteins are generally more stable than α-helices, predicting unfolding forces from sequence or structure remains challenging. This work combines force spectroscopy and molecular dynamics simulations to understand the sequence–structure–mechanical stability relationships.
Target protein domains (titin I27, ICAM-1 D4, VCAM-1 D2, and Cadherin-23 EC1) were selected using MechanoProDB, a curated resource developed as part of this thesis. Sequence and structural comparisons identified a conserved motif [NE][LI][KQR]V in terminal β-strands as critical for stability, with hydrophobic residues (L, I and V) playing a key role across Immunoglobulin-like folds. Steered molecular dynamics (SMD) of wild-type and mutants revealed the contribution of the motif to mechanical stability. Atomic force microscopy (AFM) based force spectroscopy experiments were conducted on wild-type proteins domains to validate simulation results. Two Python packages, PyFMSpec and PySteMoDA, were developed to facilitate data analysis through automated processing of AFM and SMD datasets.