In vitro investigation of protein assembly by combined microscopy and infrared spectroscopy at the nanometer scale

Abstract

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca²⁺ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

Keywords: S-layer protein; nano-FTIR; operando spectroscopy; self-assembly; solid-liquid interface.

Fig. 1.

Schematic drawings of experiment, nano-FTIR spectra, and AFM topographic images of SbpA proteins forming assembly layer. (A) Schematic drawing of the nano-FTIR experiment with the AFM tip situated over the monolayer graphene suspended across 1-µm diameter holes of a perforated Si₃N₄ membrane (only one hole is shown). The membrane closes the cell filled with SbpA proteins and Ca²⁺ ions in a buffer solution. The proteins attach to the graphene membrane (colored blue) and Si₃N₄ membrane (colored green) during assembly. Free proteins in the solution are shown in gray. (B) Schematic of the high-resolution AFM imaging with the probe immersed in the same protein-buffer solution as in (A). (C) Nano-FTIR spectra of an assembled film of SbpA proteins in a buffer solution of 5 mM Ca²⁺ (black), and in 50 mM Ca²⁺(red), acquired 3 h after filling the cell (Top two graphs). The Bottom graph (blue) is an ATR-FTIR spectrum of dried SbpA proteins assembled from a 50 mM Ca²⁺ solution on a graphene-coated Au film on a Si wafer. The two nano-FTIR spectra are averages of 120–150 spectra collected in different spots of protein-covered regions. (D) AFM topographic images of the graphene membrane prior (Left) and after SbpA assembly (Right) acquired 1 h after filling the cell. The Inset is an expanded image showing the square lattice structure of the assembled proteins. The scan size is 3 µm for the large images and 90 nm for the Inset. The bright lines are due to wrinkles in the graphene. Protein domains form on both the suspended and supported regions in AFM imaging experiments where the tip scans over the suspended as well as the supported graphene. In the nano-FTIR experiments, only proteins on the suspended graphene can be observed unless damage or detachment of the graphene accidentally occurs.

Fig. 2.

Temporally resolved assembly process of SbpA protein. (A) Color map representation of the time evolution of the nano-FTIR spectra during SbpA protein assembly on the graphene membrane in a buffer solution of 5 mM Ca²⁺ (Top), and without Ca²⁺ (Bottom). The tip location was fixed during the measurement. (B) Percentage of area covered by assembled protein from AFM images in 5 mM Ca²⁺ buffer solution (black squares, Left y axis). Integrated intensity of Amide I and Amide II bands (red dots, Right y axis) from the spectra shown in the top panel of (A). The three circular insets are AFM images showing SbpA domain growth on the graphene membrane as a function of time. Time 0 corresponds to the introduction of the protein/buffer solution into the cell. The suspended graphene boundary is marked by black dashed circles.

Fig. 3.

Spatially resolved chemical mapping of SbpA protein assemblies. (A and B) Images of the total scattered optical amplitude of suspended graphene and surrounding area in the cell filled with SbpA proteins, in 5 mM Ca²⁺ buffer solution in (A) and 50 mM Ca²⁺ solution in (C). Total optical amplitude increases substantially when the probe is over the gold substrate. (B and D) Corresponding color map representations of the nano-FTIR spectral intensities (arbitrary units in the color scale) of the amide I and II mode regions of the SbpA proteins. The nano-FTIR profiles were acquired at positions along the red arrows in (A) and (C). The amide I and II modes are only visible when the tip is located over the suspended graphene region in contact with the solution. The hole boundary is marked by the horizontal dashed lines. The position of two self-assembled domains in (D) is marked by the vertical red segments. (E) Schematic drawing of the tip-sample junction showing exponential the decay of the IR near field away from the tip apex. A SbpA lattice is drawn for comparison.

Fig. 4.

Effect of solvent replacement on SbpA proteins. (A) AFM topographic image of an area containing a graphene covered hole in the cell filled with SbpA proteins after gently substituting 5 mM Ca²⁺ H₂O solution for 5 mM Ca²⁺ D₂O solution. (B) Color map representation of the nano-FTIR spectra along the line marked by the red arrow in (A). The D₂O bending mode at 1,220 cm⁻¹ is very weak indicating that D₂O did not intercalate between the protein domain and graphene. (C) AFM topographic image after a turbulent rinsing with D₂O that perturbed both the protein layers and the graphene membrane. (D) Color map representation along the direction of red arrow in (C). The appearance of an intense D₂O bending mode at 1,220 cm⁻¹, along with the weaker amide peaks in the graphene-covered hole region after the energetic rinsing, indicates that D₂O replaced H₂O and largely removed the initial protein layer covering the graphene. In addition, the D₂O peak is also visible even outside the suspended region due to intercalation between graphene and the surrounding Au film. The negative band around 1,270 cm⁻¹ (dark blue) in both cases is due to contamination of the AFM cantilever and tip with PDMS, which has a characteristic peak in that region.