Biology subjects

Milne, C.

Publications and source records attributed to Milne, C..

4 recordsLinked to original sources

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2022

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore2-14. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern15 whether this experimental approach16 allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions17. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics15) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments16 such that biologically relevant insight emerges.

biophysics↗

bioRxiv2022

Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we captured the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

molecular biology↗

bioRxiv2019

Brain-to-brain synchrony predicts long-term memory retention more accurately than individual brain measures

Little is known about the brain mechanisms that underpin how humans learn while interacting with one another in ecologically-valid environments (1-3). This is because cognitive neuroscientists typically measure one participant at a time in a highly constrained environment (e.g., inside a brain scanner). In the past few years, researchers have begun comparing brain responses across individuals (4-6) demonstrating that brain-to-brain synchrony can predict subsequent memory retention (7-9). Yet previous research has been constrained to non-interacting individuals. Surprisingly, the one study that was conducted in a group setting found that brain synchrony between students in a classroom predicted how engaged the students were, but not how much information they retained (10). This is unexpected because brain-to-brain synchrony is hypothesized to be driven, at least partially, by shared attention (11, 12), and shared attention has been shown to affect subsequent memory (13). Here we used EEG to simultaneously record brain activity from groups of four students and a teacher in a simulated classroom to investigate whether brain-to-brain synchrony, both between students and between the students and the teacher, can predict learning outcomes (Fig. 1A). We found that brain-to-brain synchrony in the Alpha band (8-12Hz) predicted students delayed memory retention. Further, moment-to-moment variation in alpha-band brain-to-brain synchrony discriminated between content that was retained or forgotten. Whereas student-to-student brain synchrony best predicted delayed memory retention at a zero time lag, student-to-teacher brain synchrony best predicted memory retention when adjusting for a [~]200 millisecond lag in the students brain activity relative to the teachers brain activity. These findings provide key new evidence for the importance of brain data collected simultaneously from groups of individuals in ecologically-valid settings.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC=\"FIGDIR/small/644047v2_fig1.gif\" ALT=\"Figure 1\">\nView larger version (43K):\norg.highwire.dtl.DTLVardef@21b335org.highwire.dtl.DTLVardef@7c9823org.highwire.dtl.DTLVardef@1e84b1dorg.highwire.dtl.DTLVardef@19b6f55_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Experimental setup and timeline. (A) Four students and a teacher were concurrently measured with EEG during a science lesson; (B) The lesson comprised four mini-lectures, each followed by a post-test. Pre-test and delayed post-tests were administered one week prior to and one week following the EEG recording session.\n\nC_FIG HighlightsO_LIElectroencephalogram (EEG) was concurrently recorded in a simulated classroom from groups of four students and a teacher.\nC_LIO_LIAlpha-band (8-12Hz) brain-to-brain synchrony predicted students performance in a delayed post-test.\nC_LIO_LIMoment-to-moment variation in alpha-band brain-to-brain synchrony indicated what specific information was retained by students.\nC_LIO_LIWhereas student-to-student brain synchrony best predicted learning at a zero time lag, student-to-teacher brain synchrony best predicted learning when adjusting for a [~]200 millisecond lag in the students brain activity relative to the teachers brain activity.\nC_LI

neuroscience↗