Mechanically unfolding the small, topologically simple protein L

Abstract

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

FIGURE 1

Three-dimensional structure and topology of protein L. (a) The structure of protein L showing the central α-helix packed against a four stranded β-sheet. The figure was generated using PDB file 1HZ6 (21), MolScript (65), and Raster3D (66). (b) Topology diagram of protein L. β-strands are shown as arrows and the helix as a rectangle. When extended in the geometry shown (black solid arrows), the parallel terminal β-strands (shaded arrows) are subjected to a shear force. Interstrand hydrogen bonds calculated to be ≤−0.5 kcal mol⁻¹ using DSSP (67) are shown as dashed arrows and point toward the acceptor. Strands are labeled I–IV in each representation.

FIGURE 2

Force-extension profiles of (protein L)₅. Force-extension profiles shown were recorded at tip retraction rates of (a) 77, (b) 230, (c) 700, and (d) 2100 nm s⁻¹. The second to fifth peaks for each unfolding series, together with the final extension of the fully unfolded polymer, are fitted with a wormlike chain model (28) for polymer elasticity (shaded line) with p = 0.4 nm.

FIGURE 3

(a) Speed dependence of the unfolding forces of (protein L)₅ (▴), (I27*)₅ (□), and (ubiquitin)₉ (•). Error bars, where shown, represent ±SE of triplicate data sets. Solid lines through each data set are a best fit to guide the eye. Data for I27 and ubiquitin taken from Brockwell et al. (23) and Carrion-Vazquez et al. (3), respectively. Fitting the data for protein L to an analytical solution (dashed line, see Materials and Methods) estimates that the height and the position of the unfolding barrier relative to the native state is smaller and shorter () than that obtained for (I27*)₅ ( (23)). Monte Carlo simulations, using the best fit parameters for protein L obtained above, give identical modal values (cross-hairs) to those predicted by the analytical model. (b) Error analysis of parameter pairs reveals degeneracy in the fit of and x_u to the observed experimental data for (protein L)₅. Contour lines link parameter pairs calculated to have equal χ² error. (c) The three experimental force frequency distributions at 1400 nm s⁻¹ are consistent with those predicted by the analytical model (dotted lines) and Monte Carlo simulation (solid black line) using the parameter pair marked by a solid circle in b.

FIGURE 4

Loading rate dependence of the unfolding force of (protein L)₅. The force at which a domain unfolds is plotted against the instantaneous loading rate at the unfolding point for each domain. Symbols show that the instantaneous loading rate differs significantly for domains extended at the same extension rate (open circles, 40 nm s⁻¹; shaded squares, 77 nm s⁻¹; open triangles, 140 nm s⁻¹; shaded upside-down triangles, 230 nm s⁻¹; open diamonds, 400 nm s⁻¹; shaded hexagons, 700 nm s⁻¹; open squares, 1400 nm s⁻¹; and shaded circles, 2100 nm s⁻¹). Solid black line joins points averaged in force and loading rate for each pulling speed. The apparent loading rate (dashed lines), calculated by multiplying the cantilever spring constant by the extension rate (40, 77, 140, 230, 400, 700, 1400, and 2100 nm s⁻¹) and measured loading rate for each retraction speed differ significantly since the protein polymer is more compliant than the cantilever.

FIGURE 5

Constant velocity molecular dynamics simulations of protein L unfolding reveal an unusually steep and narrow response to the extension of its termini. The production of very similar force-extension profiles at the same extension rate (shaded lines) suggests that protein L unfolds via a narrow bottleneck in the energy landscape. (Inset) Comparison of the initial structure (a) and structures before (b) and after (c) the force maximum (filled circles) shows that unfolding occurs when the C-terminal β-hairpin is pulled away from the rest of the structure. In this figure, simulations were carried out at 4 × 10⁹ nm s⁻¹. For clarity, every 40th data point has been plotted.

FIGURE 6

Constant force molecular dynamics simulations of protein L and I27. (a) Replicate simulations of extension of protein L at a constant force of 400 pN are shown and demonstrate that a metastable state very similar to the native state is populated before unfolding occurs in a two-state process. For clarity, every 500th data point is plotted. (b) Simulations of I27 unfolding at 400 pN show that this protein populates a metastable state for shorter periods and unfolds in a multistep manner.

FIGURE 7

Contour plot showing the difference in distance between every pair of amino acids in protein L at a total extension (protein and cantilever) of 1.6 Å before and 1.6 Å after the mechanical unfolding event. Residue numbers (left-hand side and bottom) are shown opposite cartoons (right-hand side and top) depicting the type of secondary-structure element that each residue occupies in the native state (rectangle, α-helix; arrow, β-strand). Strands are labeled I–IV and turn 1 and turn 2 are shown as T1 and T2, respectively. Pairs of residues that move farther apart from each other during unfolding are colored purple to green (−10 to 0 Å); those that become closer to one another are shown green to red (0 to 10 Å).

FIGURE 8

Contact map of protein L (bottom left) and ubiquitin (top right). Side-chain contacts (nearest distance between atoms of two residues <5 Å, calculated by CSU software (68)) made by pairs of amino acids within structural unit 1 (β-hairpin 1 and the helix) or within structural unit 2 (β-hairpin 2) are shown by green and red squares, respectively. Contacts made between these structural units are shown in black. β-strands (labeled I to IV as in Fig. 1) and α-helices, predicted by DSSP (67), are shown as arrows and rectangles, respectively, alongside each contact map. The two structural units are colored green (unit 1) and red (unit 2) in each protein and are also shown superimposed onto the three-dimensional structure of protein L (left) and ubiquitin (right).