BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered

Model-free decomposition of transient absorption spectra into components with time-dependent shape

Disentangling overlapping spectral signatures with time-dependent shape is performed using additional information contained in ultrafast transient absorption data, without applying any assumption on the underlying kinetics

Model-free decomposition of transient absorption spectra into components with time-dependent shape

Disentangling overlapping spectral signatures with time-dependent shape is performed using additional information contained in ultrafast transient absorption data, without applying any assumption on the underlying kinetics

Model-free Investigation of Ultrafast Bimolecular Chemical Reactions: Bimolecular Photo Induced Electron Transfer

Using photoinduced bimolecular electron transfer reactions as example we demonstrate how diffusion controlled bimolecular chemical reactions can be studied in a model-free manner by quantitatively combining different ultrafast spectroscopical tools

Understanding artifacts in chiroptical spectroscopy

Spectroscopic techniques can extract information about the chiral structure of molecules. However, because these methods typically rely on chiroptical effects that are very weak, even small errors in our control of optical polarization can induce experimental artifacts. Distinguishing these artifacts from true signals remains an important practical problem. Here, we present a comprehensive study of chiroptical artifacts using Raman optical activity (ROA) as a challenging example. ROA measures the difference in Raman scattering between right- and left-circularly polarized light. While ROA spectra can yield valuable information about the vibrational modes and handedness of a chiral molecule, ROA is particularly prone to artifacts as signals are 103−104 times smaller than in standard Raman spectroscopy. We develop a Mueller-matrix model to examine the origins of artifacts in ROA. We then combine our model with experimental examples from multiple ROA instruments to understand real-world artifacts. For example, we disprove a commonly held belief that ROA spectra that exhibit mirror symmetry about the intensity axis for an enantiomeric pair confirm a true signal. Based on our findings, we describe characteristics of common artifacts and propose a list of standard controls that researchers should perform to increase the likelihood that their data represent true signal. This work is intended as a resource for those working in chiroptical spectroscopy, providing methods to understand, identify, and avoid experimental artifacts