1 research outputs found
2D-IR Spectroscopy of an AHA Labeled Photoswitchable PDZ2 Domain
We 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. Only one mutant revealed a
measurable 2D IR difference signal. In contrast to the commonly observed
frequency shifts that report on the degree of solvation, in this case
we observe an <i>intensity</i> change. To understand this
spectral response, we performed classical MD simulations, evaluating
local contacts of the AHA labels to water molecules and protein side
chains and calculating the vibrational frequency on the basis of an
electrostatic model. Although these simulations revealed in part significant
and complex changes of the number of intraprotein and water contacts
upon <i>trans–cis</i> photoisomerization, they could
not provide a clear explanation of why this one label would stick
out. Subsequent quantum-chemistry calculations suggest that the response
is the result of an electronic interaction involving charge transfer
of the azido group with sulfonate groups from the photoswitch. To
the best of our knowledge, such an effect has not been described before