Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

X-ray dose-dependent structural changes of the [2Fe-2S] ferredoxin from Chlamydomonas reinhardtii.

Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.

Photochemical regeneration of flavoenzymes - An Old Yellow Enzyme case-study.

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Engineering Adenylate Cyclase Activated by Near-Infrared Window Light for Mammalian Optogenetic Applications.

Light in the near-infrared optical window (NIRW) penetrates deep through mammalian tissues, including the skull and brain tissue. Here we engineered an adenylate cyclase (AC) activated by NIRW light (NIRW-AC) and suitable for mammalian applications. To accomplish this goal, we constructed fusions of several bacteriophytochrome photosensory and bacterial AC modules using guidelines for designing chimeric homodimeric bacteriophytochromes. One engineered NIRW-AC, designated IlaM5, has significantly higher activity at 37 °C, is better expressed in mammalian cells, and can mediate cAMP-dependent photoactivation of gene expression in mammalian cells, in favorable contrast to the NIRW-ACs engineered earlier. The ilaM5 gene expressed from an AAV vector was delivered into the ventral basal thalamus region of the mouse brain, resulting in the light-controlled suppression of the cAMP-dependent wave pattern of the sleeping brain known as spindle oscillations. Reversible spindle oscillation suppression was observed in sleeping mice exposed to light from an external light source. This study confirms the robustness of principles of homodimeric bacteriophytochrome engineering, describes a NIRW-AC suitable for mammalian optogenetic applications, and demonstrates the feasibility of controlling brain activity via NIRW-ACs using transcranial irradiation.

A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission.

Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.

Wavelength-Dependent Exciton-Vibrational Coupling in the Water-Soluble Chlorophyll Binding Protein Revealed by Multilevel Theory of Difference Fluorescence Line-Narrowing.

One of the most powerful line-narrowing techniques used to unravel the homogeneous lineshapes of inhomogeneously broadened systems is difference fluorescence line-narrowing spectroscopy. When this spectroscopy was applied to multichromophoric systems so far, the spectra were analyzed by an effective two-level system approach, composed of the electronic ground state and the lowest exciton state. An effective Huang-Rhys factor was assigned for the coupling of this state to the vibrations. Here, we extend this approach by including a multilevel line shape theory, which takes into account the excitonic coupling between pigments and thereby the effect of the delocalization of the excited states explicitly. In this way, it becomes possible to extract the spectral density of the local exciton-vibrational coupling. The theory is applied to the recombinant water-soluble chlorophyll binding protein reconstituted with chlorophyll a or b and reveals a significant decrease of the Huang-Rhys factor of the local exciton-vibrational coupling with decreasing transition energy of the chlorophylls. This decrease could be due to the increase in steric interactions reducing the flexibility of the environment and red-shifting the site energy of the pigments.

Light-assisted drying for protein stabilization.

A light-based processing method to create an amorphous trehalose matrix for the stabilization of proteins is discussed. This method has potential applications in the stabilization of protein-based therapeutics and diagnostics. During light-assisted drying (LAD), proteins suspended in a trehalose solution are dehydrated using near-infrared (NIR) laser light. The goal of this study was to determine processing parameters that resulted in fast processing times and low end moisture contents (EMC), while maintaining the functionality of embedded proteins. We compared the effect of changing processing wavelength, power and resulting sample temperature, and substrate material on the EMC for two NIR laser sources (1064 and 1850 nm). The 1850-nm laser resulted in the lowest EMC (0.03 ± 0.01 gH2O / gDryWeight) after 20 min of processing on glass microfiber paper. This suggests a storage temperature of 68.3°C. We also tested the functionality of a model protein, lysozyme, after LAD processing using a standard assay. LAD showed no significant effect on the functionality of lysozyme when processed at a maximum temperature of ∼44 ° C to an EMC of 0.17 ± 0.06 gH2O / gDryWeight. LAD is a promising technique for forming amorphous trehalose solids that could stabilize proteins at ambient temperatures.

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy.

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.

Biophysical evaluation of milk-clotting enzymes processed by high pressure.

High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212MPa/5min/10°C), calf rennet (280MPa/20min/25°C), bovine rennet (222MPa/5min/23°C), and porcine pepsin (50MPa/5min/20°C) and under inactivation conditions for all enzymes (600MPa/10min/25°C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.

Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.

Optocontrol of glutamate receptor activity by single side-chain photoisomerization.

Engineering light-sensitivity into proteins has wide ranging applications in molecular studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology.

In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states.

During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2 to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2 reduction was found in a double conformation: Glu451a (∼7 Šfrom the TNC) and Glu451b (∼4.5 Šfrom the TNC). A positive peak of electron density above 3.5σ in an Fo - Fc map for Glu451a O(ℇ2) indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-E,anti Chromophore in the Photoproduct.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹5N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.

Photo-triggered fluorescent labelling of recombinant proteins in live cells.

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed. The approach is based on photo-caged split-intein mediated conditional protein trans-splicing reaction and enabled background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome - protonated and nonprotonated flavin radical-states.

Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. Using time-resolved and steady-state optical spectroscopy, we studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a microsecond to minutes timescale could be extracted for the wild-type protein using global analysis. The wild-type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD(•-) state with a very positive midpoint potential of ~ +125 mV, although no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited in a W342F mutant. The investigation of another mutation close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a timescale of several seconds. The obtained data are critically discussed with the ones obtained from another protein, Escherichia coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical but not to significantly modulate the kinetics of any light-inducted reactions.

Photoinduced formation of flavin radicals in BLUF domains lacking the central glutamine.

Blue light receptors using FAD (BLUFs) facilitate blue light-induced signal transduction via light-induced rearrangement of hydrogen bonds between the flavin chromophore and a conserved glutamine side chain. Here, we investigated the photochemistry of the BLUF domain Slr1694 from Synechocystis sp. in which the glutamine side chain was removed. Without the glutamine, no red-shifted signaling state is formed, but light-induced proton-coupled electron transfer between protein and flavin takes place similarly as for the wild-type protein. However, the lifetime of the neutral flavin semiquinone-tyrosyl radical pair is greatly prolonged from < 100 ps to several nanoseconds, which indicates that the formation of radical intermediates drives the hydrogen bond rearrangement in BLUF photoactivation. Moreover, glutamine plays a central role in the molecular organization of the hydrogen bond network in the flavin-binding pocket, as its removal enhances electron transfer from tyrosine to the excited flavin, and enables competing electron transfer from a nearby tryptophan.