Laser-enabled technique measures protein folding in water at the nanoscale
Researchers at the National Institute of Standards and Technology (NIST; Gaithersburg, MD) and colleagues have measured at the nanometer scale the characteristic patterns of folds that give proteins their three-dimensional (3D) shape in water. The technique will help scientists gain insights about the behavior of biomolecules in watery environments similar to those in cells. These insights, in turn, could increase our understanding of major diseases, including Alzheimer's, that are related to "mistakes" in protein folding.
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Previously established techniques for determining the conformation of proteins, such as infrared (IR) spectroscopy, lack the fine spatial resolution to study the tiny and diverse assemblies of properly folded and misfolded proteins. In addition, these techniques don't work well in an aqueous environment because water strongly absorbs IR light, confounding the analysis. Water had also posed severe challenges for a pioneering technique, known as photothermal-induced resonance (PTIR), that recently enabled researchers to examine peptide structure and conformation in air with nanoscale resolution.
Schematic of the setup for photo-thermal induced resonance (PTIR), which includes an infrared laser source and atomic force microscope (AFM) cantilever with a sharp tip that touches the sample and vibrates in response to the sample's light-induced expansion. PTIR can determine the folding pattern (called for example α-helix, β-sheet) of peptides (amino acid chains) in water with nanometer-scale resolution. (Image credit: NIST)
The researchers have demonstrated that PTIR can be adapted to obtain conformational structure at the nanoscale in water using two chemically similar peptides known as diphenylalanine and Boc-diphenylalanine. Diphenylalanine is related to beta-amyloid, a sticky, larger peptide linked to Alzheimer's disease.
PTIR determines the chemical composition of materials with nanoscale resolution by combining an atomic force microscope (AFM) with light from an IR laser that operates over a range of wavelengths. The characteristic wavelengths of IR light that are absorbed by the sample are akin to a molecular fingerprint, revealing its chemical composition. At each site on the sample where IR is absorbed, the material heats up, causing it to rapidly, but ever so slightly, expand. The expansion is detected, with the sharp tip of the AFM protruding from a cantilever, which oscillates like a diving board each time the sample expands. The more light that is absorbed by the sample, the greater its expansion and the larger the strength, or amplitude, of the oscillations.
As good as PTIR is, using the method in a water environment is problematic. Water strongly absorbs IR light, producing an absorption signal that can interfere with efforts to discern the sample's chemical structure. In addition, the drag force exerted by water is much stronger than in air and it typically weakens the PTIR signal, as it strongly damps the oscillations of the AFM's cantilever.
To limit water's absorption of IR light, the team placed a prism between the laser and the sample. The prism served to confine the IR light to the sample's surface, minimizing the amount that could leak out and interact with the water. To address the damping problem, the team used a laser that could operate at frequencies up to 2000 kHz. That enabled the researchers to match the frequency of the laser pulses to one of the higher frequencies at which the cantilever oscillates. The frequency matching enhanced the amplitude of the cantilever's oscillations, partially offsetting the damping due to water.
Atomic force microscope image showing topography (top) and PTIR absorption image indicating composition and conformation (bottom) of a diphenylalanine peptide fibril in water. Additional data, recording the spectrum of infrared radiation absorbed by the peptide fibrils, provides information on their folding pattern. The PTIR spectrum indicated, for example, that diphenylalanine assumes a pure anti-parallel β-sheet conformation. (Image credit: NIST)
To demonstrate the accuracy of their method, the research team compared PTIR measurements of diphenylalanine and other peptide samples in two environments: water and air. (The peptides folded similarly in both mediums, making it easier to perform the comparison.) Remarkably, the scientists achieved similar spatial resolution and contrast in water and air, demonstrating that measurements in a water environment can be performed accurately, revealing the precise conformation of peptides with nanoscale resolution.
Full details appear in the journal ACS Nano.