Researchers at the University of Twente have developed an optical method that tracks the shape changes of a single protein in liquid, without attaching anything to it. The optical method determines the protein’s structure based on its own molecular vibrations, without the labels or markers required by other methods. The method can help researchers study how proteins react to medicines, toxins and other biomolecules.
Proteins are often depicted as fixed 3D structures, with a single shape per molecule. In reality, they are constantly in motion. They bend, unfold, refold and change shape whilst transporting drugs, binding to other molecules and regulating biological processes. These movements are essential for life, and they are difficult to observe directly, particularly in the case of a single protein moving freely through a liquid. Observing a single protein usually means immobilising it or attaching a label to it, and both of these can alter its behaviour.
Engineered metasurface
Every molecule vibrates in its own distinctive way. Raman spectroscopy can measure these vibrations, thereby revealing chemical and structural information. The signal from a single protein is normally far too weak to measure. The team solved this problem using a metasurface, a specially designed surface consisting of densely packed gold nanoparticles on a gold film.
Optical method
When light strikes the surface, it is concentrated into tiny areas with a highly concentrated electromagnetic field. A protein moving through such an area emits a Raman signal ten million times stronger. From this signal, the researchers were able to determine the secondary structure of the protein and distinguish between α-helices, β-sheets, β-turns and random coil structures.
Natural behaviour
The researchers did not need to attach a fluorescent label, a chemical marker or a mechanical anchor to the protein. This ensures that its natural behaviour remains intact. They tested the optical method on bovine serum albumin, a commonly used model for human serum albumin, the most important transport protein in the blood. Albumin transports fatty acids, hormones and many medicines.
Mapping the energy landscape
Because the measurements were taken one protein at a time, the team were able to look beyond average structures. They reconstructed free-energy landscapes, which show which conformations a protein most frequently adopts and which are rarer but still possible. They also mapped the pathways between the different conformations. The most significant transitions occurred between the α-helix and β-sheet, and between the α-helix and random coil.
“Our optical method provides insight into the dynamic behaviour of individual proteins under near-physiological conditions,” says Femi Ojambati, who led the research. “This is important because it allows us to learn more about a protein’s function from the perspective of its energy landscape and also from the pathways between structures.”
A tug-of-war over shape
The team then investigated how the chemical environment controls the shape of a protein. They introduced methyl, carboxylate and amine groups onto the metasurface – groups that are common in biological and pharmaceutical molecules. They also varied the pH of the solution from acidic to neutral and basic. Electrostatic forces determined the balance. At a neutral pH, the natural α-helix conformation usually prevails. Under other conditions, the protein tends towards β-sheet or random-coil conformations. Not only the protein sequence, but also the environment determined which conformation it adopted.
Towards drug trials targeting a single protein
The technique could provide greater insight into the (incorrect) folding and aggregation of proteins. Misfolding and aggregation play a role in diseases such as Alzheimer’s, Parkinson’s and type 2 diabetes. The researchers see the platform as a step towards label-free biophysics at the single-protein level, in realistic liquid environments. With improved metasurface designs, it may be possible to capture even faster changes and extend the approach to larger biomolecules, such as nucleic acids, carbohydrates and protein complexes.
About the survey
The article ‘Label-free single protein dynamics revealed by metasurface-enhanced Raman spectroscopy’ has been accepted for publication in ACS Nano. The authors are MohammadReza Aghdaee, Sharif Zaidouni, Yeganeh Bahiraie, Anupa Kumari and Oluwafemi S. Ojambati. The research was carried out at the Department of Applied Nanophotonics, Faculty of Applied Sciences, and the MESA+ Institute for Nanotechnology at the University of Twente.
Source and photo: UTwente
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