Biology subjects

Gorel, A.

Publications and source records attributed to Gorel, A..

3 recordsLinked to original sources

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↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

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↗