External contributions, such as interface interaction between neighboring protein domains, may arise in multimeric proteins ( 25). One possible concern is whether the individual unfolding events truly reflect the properties of individual, nonperturbed GFP molecules. Additional evidence showing the single-molecule nature of our data is provided in the supporting information. It becomes obvious that each peak reflects the forced fracture of a single GFP structure along the direction defined by the linking amino acids, followed by elongation of the lengthened polypeptide chain. The spacing between the WLC curves corresponds to the gain in contour length expected from the unfolding of a single GFP molecule along the respective linkage geometry ( 16). 2 b are overlaid with a lattice of calculated worm-like chain (WLC) curves. Full-scale traces and additional data are provided in the supporting information, which is published on the PNAS web site. 2 b displays typical force-extension traces recorded with each of the five differently linked GFP polymers. Because of inversion symmetry in the geometry of force application, this freedom in the molecular construction has no influence on force-extension data. Double-cysteine-based polymerization does not control the direction of inclusion of individual modules in the polymerized protein chain.
2 a) when single polyprotein chains are stretched between cantilever tip and surface of an atomic force microscope.
The spatial positions of the linking cysteines in the folded tertiary structure define the points of force application to the molecule ( Fig. By introducing two cysteine residues at the desired linkage points, we constructed disulfide-linked polyprotein chains ( 16) with five particular linkage geometries (see Fig. Surprisingly, previous experiments loading single GFP molecules in the conventional N- to C-terminal direction revealed very low mechanical stability ( 22) compared with other β-sheet protein structures, like the muscle protein titin ( 23, 24). The β-barrel structure of the GFP-like fold ( 18) is commonly considered extremely stable ( 19– 21). In the current study, we chose GFP to explore the three-dimensional deformation response and mechanical stability of a prominent protein structure motif. In this study, we employ cysteine engineering ( 16) to gain precise control over the points of force application to a single protein structure. 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). 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). 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. Current recombinant protein expression naturally links individual protein modules by their N and C termini.
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). Many 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). 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. From potential widths we estimated directional spring constants of the GFP structure and found values ranging from 1 N/m up to 17 N/m. We show that straining the GFP structure in one of the five directions induces partial fracture of the protein into a half-folded intermediate structure. We found fracture forces widely varying from 100 pN up to 600 pN. We investigated the deformation response of GFP in five selected directions. 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. So far, access has been limited to mostly one spatial direction of force application. Single-molecule methods have given experimental access to the mechanical properties of single protein molecules.
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