Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling

Femtosecond Stimulated Raman Study of the Photoactive Flavoprotein AppABLUF

Femtosecond stimulated Raman Spectroscopy (FSRS) is applied to study the photocycle of a blue light using flavin (BLUF) domain photoreceptor, AppABLUF. It is shown that FSRS spectra are sensitive to the light adapted state of the protein and probe its excited state dynamics. The dominant contribution to the most sensitive excited state Raman active modes is from flavin ring modes. However, TD-DFT calculations for excited state structures indicate that reproduction and assignment of the experimentally observed spectral shift will require high level calculations on the flavin in its specific protein environment

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

Influencing factors of observed speed and rule compliance of speed-pedelec riders in high volume cycling areas : Implications for safety and legislation

Speed pedelecs (s-pedelecs) are electric bicycles offering pedal assistance up to 45 km/h. S-pedelecs may contribute to a more efficient and green traffic system. However, their potential to reach high speeds has raised road safety concerns. In the Netherlands a new legislation bans s-pedelecs from bicycle paths in urban areas. On the roads with a maximum speed limit of 50 km/h with adjacent bicycle paths, s-pedelec riders must use the roadways instead of the bicycle path. The impact of this legislation on the behaviour of s-pedelec riders and other road users as well as the possible consequences for road safety are yet unknown. Therefore, this naturalistic riding study investigated the safety-relevant behaviours of s-pedelec riders, i.e. speed characteristics while riding on the roadway, the extent of non-compliance with the ban on using bicycle paths, and speed and speed adaptation while using bicycle paths. Furthermore the study explored factors possibly influencing rider behaviour (the s-pedelec's motor-power, riders’ beliefs and perceptions) as well as negative reactions of other road users encountering s-pedelec riders. 28 participants used a s-pedelec (a 350 W type or a 500 W type) for everyday trips for at least a fortnight. The s-pedelecs were equipped with two action cameras with integrated sensors and GPS. The results showed that mean speed on 50 km/h roadways was 31.8 km/h, which is far below the road's speed limit. The mean speed did not differ between s-pedelec types, but the speed distribution did. The ‘500 W riders’ travelled 31.7% of the total distance in the 41–50 km/h speed band, as compared to 6.9% of the ‘350 W riders’. Furthermore the 500 W riders evaluated riding on the roadway more positively than the 350 W riders. On the roadway s-pedelec riders experienced signals of hinderance of the traffic flow (on average every 2 km) and negative reactions from drivers (on average every 27.5 km). As for non-compliance riders covered on average 22.5% of the distance on bicycle paths. The more the riders disagreed with the new legislation, the more distance they covered on the bicycle path. Mean speed on bicycle paths was 28.5 km/h, and it was significantly higher for 500 W riders than for 350 W riders. Speeds between 41 and 50 km/h were also far more common for 500 W riders (14.9% of the distance) than for 350 W riders (0.5%). Compared to the roadway 350 W riders reduced their speed on the bicycle paths to a higher extent (from 31.4 to 25.7 km/h) than 500 W riders did (from 31.9 to 30.5 km/h). The frequency of harsh braking of s-pedelec riders was low and did not differ between the roadway and the bicycle paths. In conclusion, s-pedelec riders in the Netherlands frequently ride on the bicycle paths although it is illegal. On the bicycle paths their speeds are much higher than those of conventional cyclists. On the 50 km/h-roadways, however, s-pedelec riders are apparently too slow for the traffic conditions. Overall, the speed profiles of 350 W types were better suited to the bicycle paths, whereas those of 500 W types to the roadways.</p

Traffic conflicts involving speed-pedelecs (fast electric bicycles) : A naturalistic riding study

Speed-pedelecs -fast electric bicycles offering pedal support up to a speed of 45 km/h- are a recent, environmentally friendly, and mobility efficient innovation. However, their high travel speed may increase crash and injury risk. Due to their recent introduction accurate crash data are not available yet. Since near-crashes may serve as a proxy for crashes this study analyzed traffic conflicts (i.e., near-crashes and minor crashes) in the Netherlands with the aim to proactively identify potential crash partners, crash patterns, and crash risk increasing factors. To this end, twenty-eight participants used a speed-pedelec in daily traffic, equipped with a forward and a backward facing camera, for two to three consecutive weeks. In a total of 227 h of video footage in which a distance of 6584 km was travelled, 115 conflicts were identified of which 114 were near-crashes in which evasive actions were performed to avert a crash, and one was a minor crash. The most frequent conflict partner were bicycles (51 %), followed successively by cars and vans (28 %), pedestrians (12 %), powered two-wheelers (5 %) and animals (3 %). One conflict was with a truck. With conventional bicycles, most conflicts occurred in crossing maneuvers (36 %) and when the speed-pedelec and bicycle were travelling in the same direction (36 %). Also, with cars and vans, most conflicts occurred in crossing maneuvers (63 %). The case-cohort analyses in which characteristics in conflicts and characteristics in randomly selected moments of the same participant were identified, showed conflict risks to be high if: (1) bicycles or cars were in the proximity of the speed-pedelec but was substantially higher for bicycles than for cars (OR = 43.28, 95 % CI = [16.85−111.17] and OR = 22.43, 95 % CI = [7.59−66.28] respectively), (2) speed-pedelecs overtook other road users which were mostly bicycles (OR = 17.25, 95 % CI = [7.58−39.24]), (3) the speed-pedelecs travelled on bicycle facilities (both legally or illegally) (OR = 1.81, 95 % CI = [1.08−3.03]), and (4) speed-pedelecs rode near or at an intersection, OR = 3.94, 95 % CI = [2.42−6.43]. These findings suggest that conflict risks are higher when speed-pedelec riders make use of bicycle facilities than when they ride on the roadway for cars. However, the consequences of crashes with motorized vehicles on the roadway will probably be more severe for speed-pedelec riders than with bicycles on the cycle path. This study further illustrates the value of naturalistic conflict observations for assessing the safety implications of innovations proactively.</p

Speed characteristics of speed pedelecs, pedelecs and conventional bicycles in naturalistic urban and rural traffic conditions

To assess the potential impact of the higher speeds of pedal-assisted bicycles on safety, this study compared conventional bicycles, pedelecs and speed pedelecs (hereafter called s-pedelecs) on mean speeds, speed variability, harsh braking events (decelerations > 2 m/s), and mean speeds above the speed limit (MSAL) in rural and urban areas in the Netherlands Data were collected in daily traffic, while the legal maximum speed for speed-pedelecs was 25 km/h, and pedelecs and s-pedelecs shared the infrastructure with conventional bicycles. Data were collected, using two-wheelers equipped with accelerometers and GPS. Personality factors – sensation seeking and risk taking - were measured with surveys. Regular commuters used one of the three bicycle types for two weeks. Participant bias was intentionally included by allowing participants to select a bicycle type of their preference, resulting in 12 conventional bicycle riders (71 % women), 14 pedelec riders (67 % women) and 20 s-pedelec riders (25 % women). S-pedelecs were much faster than conventional bicycles, amounting to a speed difference with conventional bicycles of 10.4 km/h in urban areas (M =28.2 km/h vs. 17.8 km/h) and of 13.2 km/h in rural areas (M = 31.4 km/h vs. 18.2 km/h). The speed differences between pedelecs and conventional bicycles were much smaller: 2.3 km/h in urban areas (20.1 km/h vs 17.8 km/h) and 4 km/h in rural areas (22.2 km/h vs. 18.2 km/h). Compared to conventional bicycles, s-pedelecs varied their speed to a greater extent and also braked harshly more frequently, showing a greater need for speed adjustment. These adjustments were larger at higher speeds. In contrast, pedelecs did not differ from conventional bicycles on speed variation. MSAL for s-pedelec riders differed by gender. For men the MSAL was 87 % on urban sections and 91 % on rural sections. For women, the MSAL was lower, respectively 23 and 69 %. None of the personality factors were associated with speed variability, harsh braking or MSAL. However, sensation seeking was associated with higher mean speeds on all three bicycle types. To conclude, pedelecs and conventional bicycles are similar in speed patterns, whereas the speed patterns of s-pedelecs differ significantly from the former two. The safety implications are discussed.</p

Proteins in Action: Femtosecond to Millisecond Structural Dynamics of a Photoactive Flavoprotein

[Image: see text] Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The mechanism, the underlying structural dynamics, and the time scales for regulation of this response are central questions in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins, which is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been made over 10 decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) localized dynamics occur on the pico- to nanosecond time scale, while subsequent protein structural reorganization is observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which suppresses biological function is shown to ‘short circuit’ this structural relaxation pathway, suppressing the changes which occur further away from the chromophore while accelerating dynamics close to it