Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM

Abstract

Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…Comparison of this trace to the one recorded on the native protein provides information regarding the refolding extent of individual structural regions. Initial force spectroscopy experiments on subsequent unfolding and refolding of immunoglobulin domains (Rief et al 1997;Carrion-Vazquez et al 1999) resolved their folding kinetics on a time scale of hundreds of milliseconds, which were in reasonable agreement with biochemical data. Further development of the SMFS technique led to observation of folding pathways followed by single molecules, as well as detection of intermediate states and misfolded conformations of globular proteins Schwaiger et al 2004;Oberhauser et al 1999).…”

“…Until recently, the existing technology did not allow detecting subtle changes at the single molecule level. However, the rapid progress of bionanotechnology within the past two decades has provided an intensive burst of new approaches such as Forster resonance energy transfer spectroscopy, fluorescent correlation spectroscopy, and AFM allowing the assessing properties of individual macromolecules and providing fascinating insights on their dynamics (Rief et al 1997;Medina and Schwille 2002;Lipman et al 2003).…”

“…During the last decade, a novel AFM-based approach to assessing the mechanical stability of single macromolecules was applied (Lee et al 1994;Rief et al 1997;Marszalek et al 2001), which exploited an externally applied force as a denaturant. The multidomain protein titin was covalently attached to the AFM stylus with one of its peptide end, while its other end was attached to a supporting surface.…”

Calculation of the Entropy and Free Energy from Monte Carlo Simulations of a Peptide Stretched by an External Force

“…30 Also, single molecule techniques have been developed where individual molecules can be manipulated and stretched by external forces using atomic force microscopy (AFM), [31][32][33][34] for example. A well-studied case is the muscle protein titin, where force-extension profiles of the reversible unfolding of its immunoglobulin-like domains have been obtained by AFM 32,34 and optical tweezers. 35 To interpret these experiments at the atomic level, a series of steered molecular dynamics (SMD) simulations have been carried out, mostly by Schulten's group.…”

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

“…The increased probability of nucleation formation for a profile with a sawtooth pattern shows that local ordering of SELP is crucial for nucleation. Even though the local ordering is disrupted during stretching it is expected to reform quickly as the chain is detached from the tip and allowed to relax [64]. The exponential profile showed significantly increased success rate of nucleation.…”