The noncanonical amino acid azidohomoalanine (Aha) is known to be an environment-sensitive infrared probe for the site-specific investigation of protein structure and dynamics. Here, the capability of that label is explored to detect protein-ligand interactions by incorporating it in the vicinity of the binding groove of a PDZ2 domain. Circular dichroism and isothermal titration calorimetry measurements reveal that the perturbation of the protein system by mutation is negligible, with minimal influence on protein stability and binding affinity.
View Article and Find Full Text PDFWe show that it is possible to phase multidimensional infrared spectra generated by a boxcars geometry four-wave mixing spectrometer directly from the signal generated by the molecular vibration of interest, without the need for auxiliary phasing measurements. For isolated vibrations, the phase profile of the 2D response smoothly varies between fixed phase limits, allowing for a general target for phasing independent of the degree of anharmonicity exhibited between the ground and excited state. As a proof of principle, the 2D response of the ∼2155 cm thiocyanate stretch vibration of MeSCN, a system exhibiting anharmonicity such that the 0-1 and 1-2 transitions are spectrally isolated, is successfuly phased directly from the experimental spectra.
View Article and Find Full Text PDFWe explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties.
View Article and Find Full Text PDFAzidohomoalanine (Aha) is an unnatural amino acid containing an infrared active azido side chain group that can, through frequency shifts of the azido stretch vibration, act as a probe of local structure. To realize the potential of such structural probes for protein science, we have developed a two-dimensional infrared spectrometer employing fast mechanical scanning and intrinsic phasing of the resulting spectra, leading to a lower sensitivity limit of ∼100 μOD level samples. Using this approach, we quantify the biomolecular recognition between a PDZ2 domain and two Aha-mutated peptides.
View Article and Find Full Text PDF2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain.
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