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

Assessment of stationarity horizon of the heart rate

This work presents a new method using time-varying autoregressive modelling for the assessment of heart rate signals stationarity in patients before the onset of ventricular tachyarrhythmias, including comparison with a control group. A general stationarity trend is reported for all subjects, and particularly no significant change is observed before an arrhythmic event. Evaluation of the model fitting performed by a hypothesis test suggests the presence of nonlinearities. 1 Introduction Heart rate (HR) variability analysis is a well known technique to study the interaction between the autonomic nervous system and the heart sinus pacemakers. However, classical linear methods (DFT, AR modelling) relie on the assumption of stationarity. This hypothesis is not obvious since long-term HR recordings have shown strong circadian variations, suggesting a nonstationary behaviour. Numerous studies attempted to find out some particular features of the HR dynamics preceding the onset of ventricul..