Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy

Abstract

To develop a simple method for probing the physical state of surface adsorbed proteins, we adopted the force curve mode of an atomic force microscope (AFM) to extract information on the mechanical properties of surface immobilized bovine carbonic anhydrase II under native conditions and in the course of guanidinium chloride-induced denaturation. A progressive increase in the population of individually softened molecules was probed under mildly to fully denaturing conditions. The use of the approach regime of force curves gave information regarding the height and rigidity of the molecule under compressive stress, whereas use of the retracting regime of the curves gave information about the tensile characteristics of the protein. The results showed that protein molecules at the beginning of the transition region possessed slightly more flattened and significantly more softened conformations compared with that of native molecules, but were still not fully denatured, in agreement with results based on solution studies. Thus the force curve mode of an AFM was shown to be sensitive enough to provide information concerning the different physical states of single molecules of globular proteins.

Figure 1.

A schematic view of force curve operation of the AFM with a bovine carbonic anhydrase molecule sandwiched between the AFM tip and the substrate through covalent cross-linkers. Three alternative crossings of the chain in the C-terminal region leading to the knot formation are indicated with circles in black dotted lines.

Figure 2.

(A) An example of the raw output of the force curve mode of AFM operation. The tip starts from position 1 and touches the sample at position 2, from where the cantilever is pushed up to position 3. Thence, the tip reverses its movement up to position 4, and the cantilever is pulled downward to position 5 due to the presence of tensile sample. At position 5, a covalent bond yields to the tensile force, and the cantilever jumps back to its free position at 6. D, d, E, and I are, respectively, the distance between the tip and the substrate, cantilever deflection, sample extension, and the depth of compression. The total distance covered by the piezomotor under the sample stage is D = E+d. (B) The raw data above are converted to the F–E curve by plotting the tensile force, F = −k × d versus E, where k is the spring constant of the cantilever. The horizontal positions marked as H and E₀, respectively, indicate the contact positions of the tip to the protein upon theirmutual approach and the point of their final separation upon retraction. They, therefore, respectively correspond to the approximate height of the sample and the contour length of the protein, provided that the chain is stretched normal to the substrate surface.

Figure 3.

(A) The F–E curves of BCA II under native conditions with 50 mM Tris-sulfate buffer (pH 7.5) and at ~25°C. The initial low level forces in the range of 0.1–0.2 nN are considered to represent frictional resistance in the C-terminal knotted region. Insets 1 and 2 contain representative individual curves with ~15 nm and ~30 nm initial extensions, respectively. (B) F–E curve obtained in the presence of the inhibitor. Inset is a collection of representative individual curves.

Figure 4.

(A) A collection of compressive curves obtained under the same conditions. The abscissa is the distance from the end of compression, and the ordinate is the compressive force. The pink, yellow, and cyan lines in the figure are best-fitting curves based on Equation 1 (Hertz model), Equation 2 (modified Hertz), and Equation 3′ (Tatara model), respectively. (B) Four possible cases of protein compression. In each figure, the solid and dotted lines, respectively, represent relative positions of the tip, sample, and the substrate before and after compression. Symbols correspond to those defined in the text. Case 1 indicates a small deformation from both top and bottom side of a spherical sample; case 2, a small deformation from the top only (original Hertz model) of a mushroom shaped sample; case 3, a large deformation from the top and bottom (applied for the native protein) allowing lateral extension (indicated with horizontal arrows here and in case 4); and case 4, a large deformation from the top with lateral extension (applied for denatured protein).

Figure 5.

Plot of apparent Young’s modulus of BCA II, Y_app, as a function of I_H. The abscissa is the Hertzian approach distance, I_H, defined as either I_H = I/2 for curves 1 and 2, and I_H = I for the rest of the curves, where I is the experimentally observed depth of indentation (see text for individual cases). Y_app was calculated applying Equation 2 to the experimental data. Numbers represent the following: 1, native; 2, complexed with an inhibitor; 3, in 1.0–1.5 M; 4, 2 M; 5, 3 M; and 6, 6 M GdmCl solutions.

Figure 6.

(A) F–E curves under 1.5 M GdmCl showing various degrees of resistance against tensile force in the initial extension up to 40 nm, after which chains were extended as a flexible polymer. Inset is a collection of representative individual curves. (B) Collection of compressive curves with a fitting curve based on Equation 3′ as a white line. The abscissa is the distance from the end of compression, and the ordinate is the compressive force.

Figure 7.

(A) Collection of F–E curves in 6 M GdmCl fitted with the interpolation formula (Equation 5 in text) of the WLC model in red. Inset is a collection of representative individual curves. (B) Collection of compressing curves under the same conditions with a fitting curve based on Equation 3″ as a white line. The abscissa is the distance from the end of compression, and the ordinate is the compressive force.