Anisotropic deformation response of single protein molecules

Abstract

Single-molecule methods have given experimental access to the mechanical properties of single protein molecules. So far, access has been limited to mostly one spatial direction of force application. Here, we report single-molecule experiments that explore the mechanical properties of a folded protein structure in precisely controlled directions by applying force to selected amino acid pairs. We investigated the deformation response of GFP in five selected directions. We found fracture forces widely varying from 100 pN up to 600 pN. We show that straining the GFP structure in one of the five directions induces partial fracture of the protein into a halffolded intermediate structure. From potential widths we estimated directional spring constants of the GFP structure and found values ranging from 1 N͞m up to 17 N͞m. Our results show that classical continuum mechanics and simple mechanistic models fail to describe the complex mechanics of the GFP protein structure and offer insights into the mechanical design of protein materials.cysteine engineering ͉ GFP ͉ protein mechanics ͉ protein stability ͉ single-molecule force spectroscopy M any processes in living systems, such as cell division, locomotion, and enzyme activity, depend critically on single protein molecule properties like mechanical rigidity or conformational changes (1, 2). The invention of single-molecule manipulation techniques has given experimental access to the mechanical properties of soluble protein molecules (3-8) and membrane proteins (9-12). Current recombinant protein expression naturally links individual protein modules by their N and C termini. Experimental access to the deformation response of proteins has, thus, so far been limited almost exclusively to one direction of force application: the N-to C-terminal linkage direction of polyproteins. Previous experiments with two proteins that naturally exhibit a non-N-to C-terminal linkage have indicated that the mechanics of protein structures depend on loading geometry (13,14). Theoretical studies have predicted that the deformation response of proteins may vary largely, even on the single-residue level, and that protein structures may contain ''soft'' and ''stiff'' regions (15). In this study, we employ cysteine engineering (16) to gain precise control over the points of force application to a single protein structure. Fig. 1 illustrates the quality of information that controlled force application can supply about the mechanics of a single protein. Fracture or unfolding of a protein structure proceeds on a high-dimensional energy surface determined by multiple weak interactions between amino acid residues (17). Such an energy landscape is schematically shown in Fig. 1. Conventional N-to C-terminal linkage mechanically probes only a single direction (e.g., pathway I in Fig. 1). In turn, complete control of linkage topology makes previously hidden regions of the energy landscape accessible. Directions in which the protein appears soft and fractures easily will be characterized by low-and shallowe...

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

“…The axis of force application on the molecule can be changed at will by altering the location of the protein's cysteines. The response to force of different polyprotein domains along different pulling axes has already been investigated with AFM by several groups, using diverse experimental strategies (Brockwell et al 2003;CarrionVazquez et al 2003;Dietz et al 2006a;Dietz and Rief 2006). Particularly noteworthy to this regard is the cysteine-based methodology developed by Rief group to polymerize GFP and study the response to force of this protein along five different pulling axes (Dietz et al 2006a, b).…”

“…In addition, the repeating units in these polyproteins are connected via their N-and C-termini, thus preventing the application of force on the protein along alternative axes. The ability to apply force across different domains of a molecule allows one to explore the anisotropic nature of the energy landscape of proteins (Brockwell et al 2003;Carrion-Vazquez et al 2003;Dietz et al 2006a;Dietz and Rief 2006). …”

An energetic model for macromolecules unfolding in stretching experiments

“…A hierarchy of unfolding energies of the unfolding crystals may be simply due to inhomogeneity effects of the crystal domains [14,35,56], showing variable bond-breaking barriers [33] possibly owing to interfacial energy effects [57]. Another important effect is anisotropicity of the crystals with respect to the force direction-different paths in the wiggly energy landscape lead to different unfolding energy barriers [58]-so that different unfolding forces can be induced by variable crystal orientations in the macromolecule. Another known effect, that can induce hardening, is the so-called n-effect (see [58]) that, based on statistical considerations, leading to an unfolding force growth owing to a progressively reduced number of folded crystals available for unfolding in the macromolecule for growing elongations.…”

Mechanical Properties of Bovine Rhodopsin and Bacteriorhodopsin: Possible Roles in Folding and Function

“…The data at 349 nm/s were taken from a recent work. 31 WT bacteriorhodopsin data were taken from a recent study, 26 with the number of curves at 10 nm/s (n=10), 50 …”