Biology subjects

Lane, T. J.

Publications and source records attributed to Lane, T. J..

10 recordsLinked to original sources

bioRxiv2026

The structures of two intermediates en route to DNA repair within a (6-4) photolyase

Pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are mutagenic DNA lesions produced when UV light crosslinks two adjacent pyrimidine bases. These lesions must be repaired to maintain genome integrity, a function executed by the (6-4) photolyase enzymes. This repair chemistry is complex, involving the transfer of a hydroxyl group from one base to another, accompanied or followed by cleavage of an interbase bond. It remains unclear how the enzyme orchestrates this reaction. Typically, models invoke a stepwise process in which an oxetane intermediate is transiently populated, but despite over thirty years of investigation, such an intermediate has never been definitively observed. Conducting dose-controlled X-ray crystallography on the (6-4) photolyase from Vibrio cholerae, we observed the transformation of 6-4PP into an oxetane species. Furthermore, we identified a previously unanticipated hemi-cleaved intermediate. The progressive transformation of 6-4PP into oxetane, hemi-cleaved, and fully repaired species requires a reexamination of the existing models of DNA repair by (6-4) photolyases.

biochemistry↗

bioRxiv2025

Statistical crystallography reveals an allosteric network in SARS-CoV-2 Mpro

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics with atomic resolution remains a key challenge. Here, using thousands of crystals of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the proteins correlated motions. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the correlated motions that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally linked to the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography can determine protein correlated motions and rationalize their biological function. TeaserCrystallography at scale goes beyond a single structure, revealing native-state protein dynamics.

biochemistry↗

bioRxiv2024

Denoising Reveals Low-Occupancy Populations in Protein Crystals

Advances in structural biology increasingly focus on uncovering protein dynamics and transient or weak macromolecular complexes. Such studies require modeling of low-occupancy species, for instance time-evolving intermediates and bound ligands. In protein crystallography, difference maps that compare paired perturbed and reference datasets are a powerful way to identify and aid modeling of low-occupancy species. Current methods to generate difference maps, however, rely on manually tuned parameters and, in cases of weak signal due to low occupancy, can fail to extract clear, chemically interpretable signals. We address these issues, first by showing negentropy is an effective metric to assess difference map quality and can therefore be used to automatically determine parameters needed during difference map calculation. Leveraging this, we apply total variation denoising, an image restoration technique that requires a choice of regularization parameter, to crystallographic difference maps. We show that total variation denoising improves map signal-to-noise and enables us to estimate the latent phase contribution of low-occupancy states. This technology opens new possibilities for time-resolved and ligand-screening crystallography in particular, allowing the detection of states that previously could not be resolved due to their inherently low occupancy.

biophysics↗

bioRxiv2022

Prefrontal-Parietal-cTBS effects on metacognitive awareness: real or illusory?

BackgroundNeuroimaging and lesion studies suggested that the dorsolateral prefrontal and posterior parietal cortices mediate visual metacognitive awareness. The causal evidence provided by non-invasive brain stimulation, however, is inconsistent. Objective/HypothesisHere we revisit a major figure discrimination experiment adding a new Kanizsa figure task trying to resolve whether bilateral continuous theta-burst transcranial magnetic stimulation (cTBS) over these regions affects perceptual metacognition. Specifically, we tested whether subjective visibility ratings and/or metacognitive efficiency are lower when cTBS is applied to these two regions in comparison to an active control region. MethodsA within-subjects design including three sessions spaced by one-week intervals was implemented. In each session, every participant was administered bilateral cTBS to either prefrontal, control or parietal cortices. Two concurrent tasks were performed, a real and an illusory figure task, stabilising objective performance with use of an adaptive staircase procedure. ResultsWhen performing the replicated task, cTBS was found insufficient to disrupt neither visibility ratings nor metacognitive efficiency. However, with use of Kanizsa style illusory figures, cTBS over the dorsolateral prefrontal, but not over the posterior parietal cortex, was observed to significantly diminish metacognitive efficiency. Conclusion(s)Real and illusory figure tasks demonstrated different cTBS effects. A possible explanation is the involvement of the prefrontal cortex in the creation of expectations, which is necessary for efficient metacognition. Failure to replicate previous findings for the real figure task, however, cannot be said to support, conclusively, the notion that these brain regions have a causal role in metacognitive awareness. This inconsistent finding may result from certain limitations of our study, thereby suggesting the need for yet further investigation.

neuroscience↗

bioRxiv2021

Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

biophysics↗

bioRxiv2021

Intrinsic neural activity predisposes susceptibility to a body illusion

Susceptibility to the rubber hand illusion (RHI) varies. Thus far, however, there is no consensus as regards how to explain this variation. Previous studies, focused on the role of multisensory integration, have searched for neural correlates of the illusion. Those studies, however, have failed to identify a sufficient set of functionally specific neural correlates. An alternative explanation of the illusion is that it results from demand characteristics, chiefly variability in the disposition to respond to imaginative suggestion: the degree to which intrinsic neural activity allows for a blurring of boundaries between self and external objects. Some evidence suggests that frontal power is one means of tracking neural instantiations of self; therefore, we hypothesized that the higher the frontal power during eyes-closed resting state, the more stable the self. As a corollary, we infer that the more stable the self, the less susceptible are participants to a blurring of boundaries--to feeling that the rubber hand belongs to them. Indeed, we found that frontal amplitude oscillations negatively correlate with susceptibility. Moreover, since and {delta} oscillations seem to be associated in pathological states that allow for a blurring of boundaries between self and external objects, we conjectured that the high frontal power observed in low-RHI participants is modulated by {delta} frequency oscillations. Indeed, we found this to be the case. Based on our findings we propose that the two explanatory frameworks might be complementary: that is, the neural correlates of multisensory integration might be necessary for the RHI, but a sufficient explanation requires investigation of variable intrinsic neural activity that acts to modulate how the brain responds to incompatible sensory stimuli. Highlights~Intrinsic frontal power negatively correlates with susceptibility to the RHI. ~Intrinsic power modulated by {delta} oscillations varies with susceptibility to the RHI. ~Sufficient explanation of RHI requires understanding of intrinsic neural dispositions that regulate the boundary between self and the external world.

neuroscience↗

bioRxiv2020

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

biophysics↗

bioRxiv2020

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

biophysics↗

bioRxiv2019

Temporal preparation and short-term temporal memory in depression

Patient suffering of Major Depressive Disorder (MDD) often complain that subjective time seems to drag with respect to physical time. This may point towards a generalized dysfunction of temporal processing in MDD. In the present study, we investigated temporal preparation in MDD. \"Temporal preparation\" refers to an increased readiness to act before an expected event; consequently, reaction time should be reduced. MDD patients and age-matched controls were required to make a saccadic eye movement between a central and an eccentric visual target after a variable duration preparatory period. We found that MDD patients produced a larger number of premature saccades, saccades initiated prior to the appearance of the expected stimulus. These saccades were not temporally controlled; instead, they seemed to reflect increased oculomotor impulsivity. In contrast, the latency of visually-guided saccades was strongly influenced by temporal preparation in controls; significantly less so, in MDD patients. This observed reduced temporal preparation in MDD was associated with a faster decay of short-term temporal memory. Moreover, in patients producing a lot of premature responses, temporal preparation to early imperative stimuli was increased. A reduction in premature saccades, however, was associated with reduced temporal preparation to late imperative stimuli.\n\nIn conclusion, reduced temporal preparation and short-term temporal memory in the oculomotor domain supports the hypothesis that temporal processing was altered in MDD patients. These observed deficits could reflect other underlying aspects of abnormal time experience in MDD.

neuroscience↗

bioRxiv2019

Intrinsic activity temporal structure reactivity to behavioural state change is correlated with depressive rumination

The brains intrinsic activity plays a fundamental role in its function. In normal conditions this activity is responsive to behavioural context, changing as an individual switches between directed tasks and task-free conditions. A key feature of such changes is the movement of the brain between corresponding critical and sub-critical states, with these dynamics supporting efficient cognitive processing. Breakdowns in processing efficiency can occur, however, in brain disorders such as depression. It was therefore hypothesised that depressive symptoms would be related to reduced intrinsic activity responsiveness to changes in behavioural state. This was tested in a mixed group of major depressive disorder patients (n = 26) and healthy participants (n = 37) by measuring intrinsic EEG activity temporal structure, quantified with detrended fluctuation analysis (DFA), in eyes-closed and eyes-open task-free states and contrasting between the conditions. The degree to which DFA values changed between the states was found to be negatively correlated with depressive symptoms. DFA values did not differ between states at all in those with high symptom levels, meaning that the brain remained in a less flexible sub-critical condition. This sub-critical condition in the eyes-closed state was further found to correlate with levels of maladaptive rumination. This may reflect a general cognitive inflexibility resulting from a lack in neural activity reactivity that may predispose people to overly engage in self-directed attention. These results provide an initial link between intrinsic activity reactivity and psychological features found in psychiatric disorders.

neuroscience↗