Radiation damage in protein crystallography

A general model is derived to describe the rate of radiation damage in protein crystals. This model and some of its special cases are tested against the Blake and Phillips data on X-irradiation effects in myoglobin crystals. The results point to a sequential process of damage and suggest an improved method for correcting diffraction data for the effects of radiation damage.     

Individual molecules of thermostable alkaline phosphatase support different catalytic rates at room temperature

Thermus thermophilus cells were grown at 70°C, lysed and the lysate subjected to single molecule alkaline phosphatase assays, using a capillary electrophoresis laser‐induced fluorescence detection‐based method. The enzyme was found to be heterogeneous with respect to catalytic rate when assayed at room temperature. Turnover numbers ranged 12‐fold, with an average of 400 ± 200 reactions/min for the 80 molecules assayed. Copyright © 2002 John Wiley & Sons, Ltd.     

Observation of unstable species in enzyme-catalyzed transformations using protein crystallography

Recent advances in rapid X-ray diffraction data collection methods, cryocrystallography, and other techniques have made it possible to visualize short-lived species in enzyme-catalyzed reactions directly at atomic resolution for a significant number of crystalline enzymes. The wide range of reaction types, intermediate lifetimes, and crystal characteristics means that different methods must be employed in each case, but there are enough examples now of successful structure determinations of normally unstable species to suggest guidelines for future investigations.     

Modeling radioprotective mechanisms in the dose effect relation at low doses and low dose rates of ionizing radiation

A new model (Random Coincidence Model--Radiation Adapted (RCM-RA)) is proposed which explains a possible pseudo threshold for stochastic radiation effects. It describes the formation of cancer in the case of multistep fixation of lesions in the critical regions of tumor associated genes such as proto-oncogenes or tumor-suppressor genes. The RCM-RA contains two different possibilities of DNA damage to complementary nucleotides. The damage may be caused either by radiation or by natural processes such as cellular radicals or thermal damage or by chemical cytotoxins. The model is based on the premise that radiation initially is bionegative, damaging organisms at their different levels of organization. The radiation, however, also induces various cellular radioprotective mechanisms which decrease the damage by natural processes. Considering both effects together, the theory explains apparent thresholds in the dose-response relation for radiation carcinogenesis without contradiction to the classical assumption that radiation is predominantly bionegative at doses typically found in occupational exposures.     

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined.Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.     

Bacterial protein production from corn cob pre-treated with NaOH at room temperature

Corn cobs were treated at room temperature with NaOH at a ratio of 100:3 (w/w), but with different volumes of water from 3 to 0.25 ml/g corn cob. The biomasses obtained from a mixture culture of Cellulomonas sp. and Bacillus subtilis under each condition were similar (5.5 to 6.0 g/l) and protein only varied between 30% and 40% (w/w biomass) or 1.9 and 2.2 g/l. Low volumes and low amounts of NaOH can therefore be used in a cost-effective manner.N. Pece is with the Cátedra de Quimica Orgánica, Universidad Nacional de Santiago del Estero, Argentina. N. Perotti and O. Molina are with the Cátedra de Microbiologia Industrial, Universidad Nacional de Tucumán, y PROIMI, Planta Piloto de Procesos Industriales Microbiológicos, Av. Belgrano y Pje Caseros, 4000 Tucumán, Argentina     

When X-rays modify the protein structure: radiation damage at work

The majority of 3D structures of macromolecules are currently determined by macromolecular crystallography, which employs the diffraction of X-rays on single crystals. However, during diffraction experiments, the X-rays can damage the protein crystals by ionization processes, especially when powerful X-ray sources at synchrotron facilities are used. This process of radiation damage generates photo-electrons that can get trapped in protein moieties. The 3D structure derived from such experiments can differ remarkably from the structure of the native molecule. Recently, the crystal structures of different oxidation states of horseradish peroxidase and nickel-containing superoxide dismutase were determined using crystallographic redox titration performed during the exposure of the crystals to the incident X-ray beam. Previous crystallographic analyses have not shown the distinct structures of the active sites associated with the redox state of the structural features of these enzymes. These new studies show that, for protein moieties that are susceptible to radiation damage and prone to reduction by photo-electrons, care is required in both the design of the diffraction experiment and the analysis and interpretation.     

Thermal enhancement of the radiation damage in the mouse foot at different heat and radiation dose: influence of thermotolerance

We studied the reaction of the mouse foot after combined X-irradiation and heat treatment. Acute reactions after heat differ from those after irradiation, however, after healing of the lesions, the same symptoms of deformity of the mouse foot remain. Prior heat treatment, 30 min at 43 degrees C, of the foot led to thermotolerance and this thermotolerance resulted in resistance to combined irradiation-heat treatments and hence to a decreased thermal enhancement of radiation effects. Resistance could be observed up to 168 h after prior heat treatment. The development of resistance to combined treatment at higher irradiation dose (15 or 20 Gy) and less severe heating was slower than at lower irradiation dose (10 Gy) and more severe heating. Thermal enhancement was confirmed to be dependent on the sequence of, and the interval between irradiation and heat treatment. When the mouse foot was made thermotolerant by prior heat treatment, thermal enhancement was always reduced, regardless of the sequence, when the combined heat and radiation treatments were given with an interval of less than 12 h. Thermotolerance led to an apparent decrease in the effective temperature employed in a combined treatment equivalent to approximately 1.0 degrees C, at temperatures above 43 degrees C in a 1 h heat treatment.     

Correlation of intensity-modulated radiation therapy at a specific radiation dose with the prognosis of nasal mucous damage after radiotherapy

Radiation and Environmental Biophysics - Objective of the present study was to investigate the tolerant radiation dose of nasal mucosa by observing and analyzing patients who received...     

Light-induced protein-matrix uncoupling and protein relaxation in dry samples of trehalose-coated MbCO at room temperature

In humid samples of trehalose-coated carboxy-myoglobin (MbCO), thermally driven conformational relaxation takes place after photodissociation of the carbon monoxide (CO) molecule at room temperature. In such samples, because of the extreme viscosity of the external matrix, photodissociated CO cannot diffuse out of the protein and explores the whole (proximal and distal side) heme pocket, experiencing averaged protein heme pocket structures, as a results of the presence of Brownian motions. At variance, in very dry samples, a lower portion of the photodissociated CO diffuses from the distal to the proximal heme pocket side probing in nonaveraged structures. We revisit here the flash photolysis data by Librizzi et al. (2002) and report on new, room temperature experiments in MbCO-trehalose samples, shortly illuminated prior the laser pulse. In dry samples, pre-illumination increased the diffusion of CO from the distal to the proximal heme pocket side, which resulted in less structure than in non-pre-illuminated samples. Such an effect, which is absent in humid samples, stems from a decoupling of the protein internal degrees of freedom from those of the external water-sugar matrix. We suggest that such a decoupling can be brought about by the continuous attempts performed by the protein during pre-illumination to undergo relaxation toward the photodissociated deoxy state. This, in turn, causes a collapse in the hydrogen bond network, which connects the protein surface to the water-sugar matrix, as reported by Cottone et al. (2002) and Giuffrida et al. (2003). In the conclusion section, we discuss the possible involvement of the processes invoked to rationalize the present data, in the function of macromolecules and interactions in living cells.     

Steady-state room temperature fluorescence and CO2 assimilation rates in intact leaves

Steady-state room temperature variable fluorescence from leaves was measured as a function of CO₂ pressure in Xanthium strumarium L. and Phaseolus vulgaris L. Measurements were made in a range of light intensities, at normal and low O₂ parital pressure and over a range of temperatures. At low CO₂ pressure fluorescence increased with increasing CO₂. At higher CO₂ pressure fluorescence usually decreased with increasing CO₂ but occasionally increased slightly. The transition CO₂ pressure between the responses could be changed by changing light, O₂ pressure, or temperature. This breakpoint in the fluorescence-CO₂ curve was a reliable indicator of the transition between ribulose 1,5-bisphosphate (RuBP) saturated assimilation and RuBP regeneration limited assimilation. The fluorescence signal was not a reliable indicator of O₂-insensitive assimilation in these C₃ species.     

Single cell protein production from bagasse pith pretreated with sodium hydroxide at room temperature

Summary Previous publications have revealed that a pretreatment of lignocellulosic wastes is necessary if they are to be employed as the hydrocarbon source of single cell protein production. A hot alkaline treatment is the most common.We have treated sugar cane bagasse pith with 1% NaOH solution at room temperature, at a NaOH/pith ratio of 10%. Different contact times were used in the experiments. The shortest contact period required for maximum protein production was 24 h at 25° C. A mixed culture of Cellulomonas sp. and Bacillus subtilis was used in the experiments. The values obtained for hemicellulose and cellulose in the treated pith did not differ greatly from those of untreated pith, in contrast the amount of lignin was 33% lower in the treated pith. The effect of reutilization of the alkaline liquor used for the pretreatment of pith upon protein production was also investigated. With four recyclings, there was a NaOH saving of 34.4 kg per 100 kg produced protein as compared to when the liquor was only used once.The quality of the resulting effluents, as measured by the chemical oxygen demand (COD), proved to be very similar for both types of treatment.     

Polarised asymmetric inheritance of accumulated protein damage in higher eukaryotes

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny.     

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the “Warburg effect”) and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this ‘glutamine addiction’ of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.     

History of protein crystallography in China

China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein crystallography in China has reached a new climax with a number of outstanding accomplishments. Here, I review the history and progress of protein crystallography in China and detail some of the recent research highlights, including the crystal structures of two membrane proteins as well as the structural genomics initiative in China.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.