Force Spectroscopy with 9-μs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry

Abstract

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 μm² cantilever into one terminated with a gold-coated, 4 × 4 μm² reflective region connected to an uncoated 2-μm-wide central shaft. This "Warhammer" geometry achieved 8.5-μs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM.

Figure 1

Improved performance of modified AFM cantilevers. (A) Schematic of the assay showing a polyprotein consisting of four domains of NuG2 (red) and one domain of α₃D (blue) being unfolded with a Warhammer cantilever. (B–E) Images of cantilevers prior to gold removal: an unmodified BioLever Mini (B), a standard Mod Mini (C), a Long-cut Mod Mini (D), and a Warhammer (E). The cantilever’s spring constant is noted below each image. (F) Comparison of the force PSD for each cantilever using the color code denoted in (B)–(E). (G) Force precision over a given averaging time, technically the Allan deviation (17). At the very shortest times, the motion of the cantilever becomes correlated, distorting the force precision calculation. This region of the curve is de-emphasized using a dashed line.

Figure 2

Temporal resolution of different cantilever geometries. (A) Scanning electron microscopy images of the modified cantilevers. (B) Force-versus-time traces show unfolding of the (NuG2)₂-α₃D-(NuG2)₂ construct. In this assay, the construct was stretched until the α₃D and three NuG2 domains unfolded. The stage was further retracted until the polyprotein was held at ∼80 pN. The stage retraction was then stopped and the last folded NuG2 domain unfolded. Data smoothed to 2 kHz. (C) High-bandwidth force-versus-time traces from (B) showing the unfolding of the fourth NuG2 domain at v = 0 nm/s. Time constants determined from exponential fits. Data acquired at 500 kHz. (D) Autocorrelation of the cantilever motion after the final NuG2 domain unfolded but was still attached to the polyprotein. Time constants shown were determined from the 1/e point of the autocorrelation (dashed line). (E) Force PSDs of the 500-kHz data after unfolding of the final NuG2 domain. Time constants estimated from τ ≈ Q/(πƒ_c) based on the characteristic frequency, ƒ_c.

Figure 3

Improved AFM-based SMFS studies of calmodulin unfolding when using a Warhammer cantilever. (A) Force-extension curves show the unfolding of a polyprotein containing calmodulin and four repeats of NuG2 at v = 100 nm/s. Trace color coding indicates fully folded calmodulin (blue), partial unfolding of the N-terminal domain (green), followed by the full unfolding of the C- and N-terminal domains (orange and purple, respectively). The unfolding of the NuG2 domains at higher force is also color coded purple. Dashed lines represent worm-like chain fits. (Inset) Calmodulin unfolding at low force. (B) Three force-versus-time traces at v = 100 nm/s highlight three-step unfolding of calmodulin. (C) Force-extension curves comparing the unfolding of calmodulin bound and unbound to MLCK at v = 400 nm/s (red and blue, respectively). Dark traces filtered to 250 Hz.