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Nano-FTIR Offers Versatility in Protein Studies

SAN SEBASTIÁN, Spain, & Berlin and Martinsried, Germany, Dec. 17, 2013 — A new technique called Fourier transform infrared nanospectroscopy (nano-FTIR) overcomes the diffraction limits of mid-infrared spectroscopy for label-free chemical and structural imaging of protein structures with high resolution (less than 30 nm) and extreme sensitivity.

The method, devised by the nanoscience research center CIC nanoGUNE in Spain, the Free University of Berlin and the company Neaspec in Martinsried, maps protein structures with 30-nm lateral resolution and sensitivity to individual protein complexes of less than one attogram (10−18 gram).

Proteins are the basic building blocks of life. Their structure not only determines the shape of all living things, but also plays a major role in many diseases. Although a variety of methods have been developed to study protein chemistry and structure, recognizing and mapping their secondary structure on the nanometer scale — or even with single-protein sensitivity — is still a major challenge.


Infrared protein nanospectroscopy. Courtesy of CIC nanoGUNE.


Nano-FTIR is an optical technique that combines scattering-type scanning near-field optical microscopy (s-SNOM) and Fourier transform infrared (FTIR) spectroscopy.

While FTIR is often used for studying the secondary structure of proteins, by itself it cannot allow them to be mapped on the nanoscale. In nano-FTIR, a sharp metallized tip is illuminated with a broadband infrared laser beam, and the backscattered light is analyzed with a specially designed Fourier transform spectrometer. With this technique, local infrared spectroscopy of proteins can be demonstrated with a spatial resolution of less than 30 nm.

“The tip acts as an antenna for infrared light and concentrates it at the very tip apex. The nanofocus at the tip apex can be thus considered as an ultrasmall infrared light source. It is so small that it only illuminates an area of about 30 × 30 nm, which is the scale of large protein complexes,” said project leader Rainer Hillenbrand, head of the Nanooptics Group at nanoGUNE.

To demonstrate Nano-FTIR's versatility in nanoscale-resolved protein spectroscopy, the researchers measured infrared spectra of single viruses, ferritin complexes, purple membranes and insulin fibrils.

"In a mixture of insulin fibrils and [a] few viruses, standard FTIR spectroscopy did not reveal the presence of the alpha-helical viruses," said team biologist Simon Poly. "By probing the protein nanostructures one by one with nano-FTIR, we could clearly identify the virus — that is, the alpha-helical structures within the beta-sheet ones."

The nano-FTIR spectra of proteins match extremely well with conventional FTIR spectra, while increasing the spatial resolution by more than 100, they said.

“We could measure infrared spectra of even single ferritin particles — these are protein complexes of only 24 proteins. The mass of one ferritin complex is extremely small, only 1 attogram, but we could clearly recognize its alpha-helical structure,” said Iban Amenabar, who performed the nanospectroscopy experiments.

The researchers also studied single insulin fibrils, which are a model system for neurodegenerative diseases. Insulin fibrils are known to have a core of beta-sheet structure, but their complete structure is still not clear.

“In nano-FTIR spectra of individual fibrils, we recognized not only beta-sheet structure, but also alpha-helical structures, which might be of relevance for fibril association,” said Alexander Bittner, leader of the Self-Assembly Group at nanoGUNE.

“We are excited about the novel possibilities that nano-FTIR offers," Hillenbrand said. "With sharper tips and improved antenna function, we also hope to obtain infrared spectra of single proteins in the future. We see manifold applications, such as studies of conformational changes in amyloid structures on the molecular level, the mapping of nanoscale protein modifications in biomedical tissue, or the label-free mapping of membrane proteins. This could lead to a new era in infrared nanobiospectroscopy.”

The work appears in Nature Communications (doi:10.1038/ncomms3890).  

For more information, visit: www.nanogune.eu/en


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