Atomic force microscopy captures length phenotypes in single proteins

Abstract

We use single-protein atomic force microscopy techniques to detect length phenotypes in an Ig module. To gain amino acid resolution, we amplify the mechanical features of a single module by engineering polyproteins composed of up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded core of the module extend 20 A per module farther than the wild-type polyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discriminate the location of the insert and measure its size with a resolution similar to that of NMR and x-ray crystallography.

Figure 1

Single-molecule AFM measurements of the contour length of engineered polyproteins. (A) Structure of the I27 Ig-like module and location of the five Gly insertion sites. The N and the C termini of I27 are antiparallel and come in close apposition over the A′G strands. A mechanical linkage is thought to be present in the A′G overlap, providing continuity of force between the N and C termini of the folded module. Rupture of this linkage exposes the amino acids that are hidden in the fold, extending the contour length of the protein. We constructed polyproteins based on the wild-type (I27) and mutant forms of the I27 module with a five-Gly insert in the FG hairpin loop at position 75 (I27∷75Gly₅), in the N terminus region at position 7 (I27∷7Gly₅), and after the C terminus at position 89 (I27∷89Gly₅). (B) Cartoon of the sequence of events during the stretching of a polyprotein engineered with identical repeats of an I27 module. Stretching the ends of the polyprotein with an atomic force microscope sequentially unfolds the protein modules, generating a saw-tooth pattern in the force-extension relationship that reveals the mechanical characteristics of the protein (C; 10–13). 1, an anchored polyprotein composed of four Ig domains. The protein is relaxed; 2, stretching this protein to near its folded contour length, L_c⁰, requires a force that is measured as a deflection of the cantilever; 3, the applied force triggers unfolding of a domain, increasing the contour length of the protein and relaxing the cantilever back to its resting position; 4, further stretching removes the slack and brings the protein to its new contour length L_c¹. (C) Idealized force-extension curve that results from stretching a polyprotein. The numbers correspond to the stages marked in B. The dashed lines are fits of the WLC model of elasticity to the force-extension curves leading up to each force peak. The increase in contour length resulting from module unfolding is calculated from the fits as Δ = L_c¹ − L_c⁰.

Figure 2

Geometrical errors in the measurements of contour length. (A) A relaxed polyprotein with a length l₀ is picked up by the AFM tip and stretched by force to a folded contour length L_c. The stretched polyprotein is pulled at an angle θ₁. Module unfolding extends the protein to a contour length L_c + Δ. However, the measured increase, Δ^m, is a function of θ₁. (B) We calculate this error as a function of θ₁ for a typical polyprotein composed of 10 identical I27 domains with l₀ = 8.6 nm; L_c = 38 nm and Δ = 280 nm. Under these conditions, the maximal error in the measurement of Δ is less than 1% and occurs at a θ₁ = 77^o. Molecules that are stretched from higher θ₁ values have less error. When θ₁ = 90°, the error is zero (see Materials and Methods).

Figure 3

Amplification by polyprotein unfolding captures small changes in the length of a mutant I27 module. Insertion of five Gly residues into the FG loop of the I27 module increases the contour length of the unfolded module by 2 nm. (A) Comparison of the force-extension curves of a wild-type I27 polyprotein (I27₁₂; black trace) and a mutant polyprotein, I27₁₀∷75Gly₅ (red trace). In both cases, domain unfolding increases the contour length of the proteins as they are extended. However, the I27₁₀∷75Gly₅ polyprotein extends more with each unfolding event. In the example shown, after 10 consecutive unfolding events the mutant polyprotein is 19.1 nm longer than the wild-type polyprotein (arrows), corresponding to a difference of 1.91 nm/module. (B and C) The saw-tooth pattern in the force-extension curves obtained from polyproteins engineered with five Gly inserts placed either in position 89 (I27₅∷89Gly₅; red trace in B) or in position 7 (I27₉∷7Gly₅; red trace in C) superimpose on the saw-tooth pattern obtained from the wild-type polyprotein (I27₁₂; black trace in B and C).

Figure 4

(A) Measurement of the contour length of an I27₁₂ protein before (L_c⁰) and after (L_c¹²) unfolding of its 12 modules. The solid lines are fits of the WLC model to the data. We measured L_c⁰ = 92 nm (P = 0.57 nm) and L_c¹²= 436 nm (P = 0.29 nm) and calculate an elongation per module of Δ = (L_c¹² − L_c⁰)/12 = 28.7 nm. The average values for the persistence length in the folded state were 0.87 ± 0.11 nm and 0.35 ± 0.02 nm in the unfolded state (n = 18). (B) Histogram of the contour length increment caused by the unfolding of a single module (Δ) measured for the wild-type and mutant polyproteins. Gaussian fits to each histogram are shown as solid lines.