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To determine the structure of biological molecules, theoretical predictions are matched to experimental data provided by cold ion laser spectroscopy

Scientists at the École Polytechnique Fédérale de Lausanne (EPFL), Switzerland, have developed a new method based on infrared and UV lasers to more accurately determine the extremely complex three-dimensional structures of proteins. The research advance is fundamental as such biological molecules often contain the same sequence of thousands of atoms, but their 3D structure can be radically different, which means the proteins behave completely differently.

To determine the structure of biological molecules, theoretical predictions are matched to experimental data provided by cold ion laser spectroscopy, a technique developed for over a decade at EPFL’s Laboratory of Molecular Physical Chemistry (LCPM) and now extended to include a new fingerprint (characteristics of a molecule that reflect its 3D structure) for greater accuracy. “The IR-UV (infrared-ultraviolet) double resonance laser spectroscopy method enables measuring IR absorption spectra of large protonated molecules, including peptides and, so far, small proteins in the gas phase,” explains LCPM’s Oleg Boyarkine, PhD. “In addition to measurements of vibrational frequencies of these species, which has been demonstrated previously, we now can measure absolute absorption intensities for such molecules.”

To do so, the ionised biomolecules are cooled to temperatures close to absolute zero, around 10 K or -263°C. The molecules are then shot with infrared and UV lasers at different energies, each producing a different number of molecular fragments. A mass spectrometer measures these fragments and provides information on the energy levels within the molecule. “One such quantity can be vibrational spectra, which reflect vibrational frequencies of nuclei in a molecule and how well these vibrations can absorb light,” Boyarkine explains. The latter, he adds, is characterised by absolute absorption intensities, which generally are very difficult to measure even for small molecules. Large biological molecules like proteins can have hundreds to thousands of frequencies that together provide a unique fingerprint of the molecule’s 3D structure. Altogether, the experimental frequencies and intensities provide a set of constraints, which then allows a strict verification of the calculated structures.

Being able to accurately determine the 3D structure of a protein in great detail can have immense significance for the fields of molecular biology, biotechnology and medicine. For instance, designing drugs requires knowledge of three-dimensional structure of a targeted protein, to which the drug should be attached. “It is like finding an appropriate key to match to a lock,” Boyarkine illustrates, stating further that the more constraints an experiment provides, the higher is the level of confidence in the accuracy and the limitation of a particular theoretical approach. “High confidence in calculated structures of biomolecules will allow avoiding mistakes in the interpretation of the mechanisms of their biological functionality,” the expert says. “It will also help in silica intelligent drug design.” EPFL’s new findings strengthen the ability of spectroscopy to determine molecular structures and extend it to larger species of protein size.

The biggest intellectual challenge, Boyarkine recounts, was to understand the mechanism of the IR-UV spectroscopic technique on a quantum-mechanical level. “Once done, we were able to advise a simple procedure for measurements of absolute absorption intensities and extend the use of CIS from polypeptides to proteins.” By deciphering how CIS works and thus being able to measure the absolute absorption intensity for each vibration of a molecule, the scientists created a whole new fingerprint to work with.

During the next phase of this research project, studying even larger biomolecules with extremely complicated 3D structures will require more experimental details. “We now plan to combine high-resolution photodissociation cold ions spectroscopy with high-resolution mass-spectrometry,” Boyarkine says. “This may allow a three-dimensional mapping of biomolecules by measuring the UV photodissociation yield in the function of UV laser wavelength and the mass of the fragments.” Such complex 3D maps could then be very specific fingerprints of biomolecules that would facilitate their unambiguous identification.

Written by Sandra Henderson, Research Editor, Novus Light Technologies Today

Labels: infrared,IR,UV,spectroscopy,3D,biophotonics,laser,biomolecules

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