Site-Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

Abstract

Real-time observation of structure change associated with protein function remains a major challenge.Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, PixD and AppABLUF; both proteins retain the red shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared (TRIR) difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins, and a differential response of the two sites to chromophore excitation.

Lessons from combined experimental and theoretical examination of the FTIR and 2D-IR spectroelectrochemistry of the amide I region of cytochrome c

“…With future improvement of experimental and simulation procedures, a simultaneous fitting based on the FTIR as well as 2D-IR spectra may be within reach. However, it may be more fruitful to employ isotope labelling methods 17,18,[62][63][64][65][66][67][68][69] or IR labels [70][71][72][73][74][75][76][77][78] to reveal information on local structural changes.…”

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

“…7,[28][29][30][31][32][33][34] A challenge is posed by the need to separate spectral contributions originating from the chromophore and the protein environment, a task that can be approached by a combination of (i) analysis of the spectra of the isolated chromophore, 35,36 (ii) isotopic labelling of key residues such as the glutamine (achieved currently only for a BLUF domain), 37 and the chromophore, 33,38 (iii) mutation of key residues 32,39,40 or (iv) by inserting non-canonical amino acid probes in the protein sequence. 41 More recently, femtosecond-stimulated Raman experiments indicate that FMN modes can be selectively enhanced under appropriate resonance conditions. 35,42,43 Computational efforts have been instrumental in the elucidation of large scale changes of LOV domains via molecular dynamics simulations.…”

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

“…In TRIR this separation has been addressed through the study of isotopically substituted flavins, by isotope editing key protein residues or by site specific introduction of IR marker modes, using noncanonical amino acid substitution. 13,[15][16][17] More recently the technique of femtosecond stimulated Raman spectroscopy (FSRS) has been developed to measure the vibrational Raman spectrum of excited electronic states and photoproducts. [18][19] In addition to its ability to record transient real-time Raman spectra, FSRS can exploit resonance enhancements to probe specifically chromophore excited states.…”