Impact of charge state on 193 nm ultraviolet photodissociation of protein complexes.

As applications in mass spectrometry continue to expand into the field of structural biology, there have been an increasing number of studies on noncovalent biological assemblies. Ensuring that protein complexes maintain native-like conformations and architectures during the transition from solution to the gas phase is a key aim. Probing composition and arrangement of subunits of multi-charged complexes via tandem mass spectrometry (MS/MS) may lead to protein unfolding and the redistribution of charges on the constituent subunits, leading to asymmetric charge partitioning and ejection of a high-charged monomer. Additionally, the overall dissociation efficiency of many ion activation methods is often suppressed for low charge states, hindering the effectiveness of MS/MS for complexes that have low charge density. Ultraviolet photodissociation (UVPD) of proteins using 193 nm photons is a high-energy alternative to collisional activation and demonstrates little to no charge state dependence. Here the symmetry of charge partitioning upon UVPD is evaluated for an array of multimeric protein complexes as a function of initial charge state. The results demonstrate that high laser energies (3 mJ) for UVPD induces more symmetric charge partitioning and ejection of low-charged, presumably compact monomers than higher-energy collisional dissociation (HCD).

UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking.

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

UVA-induced upregulation of progerin suppresses 53BP1­mediated NHEJ DSB repair in human keratinocytes via progerin-lamin A complex formation.

Ultraviolet (UV) radiation is the primary risk factor underlying photoaging and photocarcinogenesis. Mounting research has focused on the role of DNA damage response pathways in UV-induced double-strand break (DSB) repair. In the present study, we hypothesized that UVA-induced aberrant progerin upregulation may adversely affect p53-binding protein 1 (53BP1)-mediated non-homologous end joining (NHE) DSB repair in human keratinocytes. Basal cell carcinoma (BCC) tumors and matching normal skin tissue were sampled (n=200) to investigate whether human keratinocytes display dysregulated progerin expression as a function of advancing age and BCC status. Newborn foreskin samples (n=9) were used as a source for primary keratinocyte cultures. We investigated the effects of UVA radiation on progerin and lamin A expression as well as the effects of the silencing of progerin on lamin A protein expression in UVA-irradiated keratinocytes. We investigated whether blocking progerin­lamin A interaction was able to rescue UVA-induced lamin A protein downregulation, 53BP1 downregulation and 53BP1-mediated NHEJ DSB repair activity. Progerin upregulation in adult keratinocytes was associated with advancing age, not BCC status. In vitro, UVA exposure significantly upregulated progerin expression by favoring alternative LMNA gene transcript splicing. UVA exposure significantly downregulated free (unbound) lamin A protein levels via progerin-lamin A complex formation. UVA exposure significantly decreased 53BP1 protein levels via enhanced progerin-lamin A complex formation. UVA-induced progerin­lamin A complex formation was largely responsible for suppressing 53BP1-mediated NHEJ DSB repair activity. The present study is the first to demonstrate that UVA-induced progerin upregulation adversely affects 53BP1-mediated NHEJ DSB repair in human keratinocytes via progerin­lamin A complex formation.

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

Effects of gamma irradiation on collagen damage and remodeling.

PURPOSE: To evaluate the dose-time dependences of structural changes occurring in collagen within 24 hours to three months after gamma-irradiation at doses from 2-40 Gy in vivo. MATERIALS AND METHODS: Rat's tail tendon was chosen as in vivo model, with its highly ordered collagen structure allowing the changes to be interpreted unambiguously. Macromolecular level (I) was investigated by differential scanning calorimetry (DSC); fibers and bundles level (II) by laser scanning microscopy (LSM), and bulk tissue microstructural level (III) by cross-polarization optical coherence tomography (CP-OCT). RESULTS: For (I), the formation of molecular cross-links and breaks appeared to be a principal mechanism of collagen remodeling, with the cross-links number dependent on radiation dose. Changes on level (II) involved primary, secondary and tertiary bundles splitting in a day and a week after irradiation. Bulk collagen microstructure (III) demonstrated early widening of the interference fringes on CP-OCT images observed to occur in the tendon as result of this splitting. At all three levels, the observed collagen changes demonstrated complete remodeling within ∼ a month following irradiation. CONCLUSION: The time course and dose dependencies of the observed collagen changes at different levels of its hierarchy further contribute to elucidating the role of connective tissue in the radiotherapy process.

Photo-induced reversible structural transition of cationic diphenylalanine peptide self-assembly.

The photo-induced self-assembly of a cationic diphenylalanine peptide (CDP) is investigated using a photoswitchable sulfonic azobenzene as the manipulating unit. A reversible structural transition between a branched structure and a vesicle-like structure is observed by alternating between UV and visible light irradiation.

Conversion from CUL4-based COP1-SPA E3 apparatus to UVR8-COP1-SPA complexes underlies a distinct biochemical function of COP1 under UV-B.

The evolutionarily conserved constitutive photomorphogenesis 1 (COP1) is a RING and WD40 protein that functions as a substrate receptor of CULLIN4-damaged DNA binding protein 1 (CUL4-DDB1)-based E3 ubiquitin ligases in both plants and animals. In Arabidopsis, COP1 is a central repressor of photomorphogenesis in the form of COP1-suppressor of PHYA (SPA) complex(es). CUL4-DDB1-COP1-SPA suppresses the photomorphogenic program by targeting the transcription factor elongated hypocotyl 5 for degradation. Intriguingly, under photomorphogenic UV-B light, COP1 reverses its repressive role and promotes photomorphogenesis. However, the mechanism by which COP1 is functionally switched is still obscure. Here, we demonstrate that UV-B triggers the physical and functional disassociation of the COP1-SPA core complex(es) from CUL4-DDB1 and the formation of a unique complex(es) containing the UV-B receptor UV resistance locus 8 (UVR8). The establishment of this UV-B-dependent COP1 complex(es) is associated with its positive modulation of elongated hypocotyl 5 stability and activity, which sheds light on the mechanism of COP1's promotive action in UV-B-induced photomorphogenesis.

Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer.

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

Reversing the direction in a light-driven rotary molecular motor.

Biological rotary motors can alter their mechanical function by changing the direction of rotary motion. Achieving a similar reversal of direction of rotation in artificial molecular motors presents a fundamental stereochemical challenge: how to change from clockwise to anticlockwise motion without compromising the autonomous unidirectional rotary behaviour of the system. A new molecular motor with multilevel control of rotary motion is reported here, in which the direction of light-powered rotation can be reversed by base-catalysed epimerization. The key steps are deprotonation and reprotonation of the photochemically generated less-stable isomers during the 360° unidirectional rotary cycle, with complete inversion of the configuration at the stereogenic centre. The ability to change directionality is an essential step towards mechanical molecular systems with adaptive functional behaviour.

Aggregation of amyloid Abeta((1-40)) peptide in perdeuterated 2,2,2-trifluoroethanol caused by ultrasound sonication.

Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Abeta((1-40))-peptide is studied by pulsed-field gradient (PFG) spin-echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Abeta((1-40)) in perdeuterated trifluoroethanol, CF(3)-CD(2)-OD (TFE-d(3)), the peptide is present in the solution as a stable monomer adopting alpha-helical secondary structural motifs. The self-diffusion coefficient of Abeta((1-40)) monomers in TFE-d(3) was measured as 1.35 x 10(-10) m(2) s(-1), reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of Abeta molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 x 10(-13) m(2) s(-1). Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in (1)H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Abeta((1-40)) in TFE-d(3). Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt alpha-helical motifs in both monomers and aggregates formed upon sonication.

Room-temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay.

The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X-ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4-benzoquinone and 2,2,6,6-tetramethyl-4-piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4-benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH* radicals (k(OH) = 8 x 10(9) M(-1) s(-1)) and electrons with a lower rate constant (k(e-(aq)) = 3.0 x 10(8) M(-1) s(-1)), doubled the crystal dose tolerance, whereas 1,4-benzoquinone, which also scavenges both OH* radicals (k(OH) = 1.2 x 10(9) M(-1) s(-1)) and electrons (k(e-(aq)) = 1.2 x 10(10) M(-1) s(-1)), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first-order to a zeroth-order process.

Mini-beam collimator enables microcrystallography experiments on standard beamlines.

The high-brilliance X-ray beams from undulator sources at third-generation synchrotron facilities are excellent tools for solving crystal structures of important and challenging biological macromolecules and complexes. However, many of the most important structural targets yield crystals that are too small or too inhomogeneous for a ;standard' beam from an undulator source, approximately 25-50 microm (FWHM) in the vertical and 50-100 microm in the horizontal direction. Although many synchrotron facilities have microfocus beamlines for other applications, this capability for macromolecular crystallography was pioneered at ID-13 of the ESRF. The National Institute of General Medical Sciences and National Cancer Institute Collaborative Access Team (GM/CA-CAT) dual canted undulator beamlines at the APS deliver high-intensity focused beams with a minimum focal size of 20 microm x 65 microm at the sample position. To meet growing user demand for beams to study samples of 10 microm or less, a ;mini-beam' apparatus was developed that conditions the focused beam to either 5 microm or 10 microm (FWHM) diameter with high intensity. The mini-beam has a symmetric Gaussian shape in both the horizontal and vertical directions, and reduces the vertical divergence of the focused beam by 25%. Significant reduction in background was achieved by implementation of both forward- and back-scatter guards. A unique triple-collimator apparatus, which has been in routine use on both undulator beamlines since February 2008, allows users to rapidly interchange the focused beam and conditioned mini-beams of two sizes with a single mouse click. The device and the beam are stable over many hours of routine operation. The rapid-exchange capability has greatly facilitated sample screening and resulted in several structures that could not have been obtained with the larger focused beam.

Focusing of macromolecules using equilibrium between centrifugal and electric force.

A focusing separation model for macromolecules has been theoretically investigated. The method involves an ultracentrifugation device, which however, deploys an electric field gradient oriented longitudinally along the radial direction. When a macromolecular sample solution is centrifuged, the molecules which have different density to the surrounding solvent and a non-zero electric charge, experience a combination of centrifugal and electric forces. This forces the molecules to move to the equilibrium positions along the radius of the rotor, which are characterised by the charge over mass ratio of the molecules. Therefore a molecular sample will be separated into its constituents and bands will form, akin to electrophoresis. The bands are, however, focused at their equilibrium position. An example configuration has been examined whereby four proteins with masses between 20 and 100 kDa can be separated within a radial distance of 20 cm, for a rotor spinning at approximately 130,000 rpm and with a varying electric field between 0 and 100 V/cm.

Tracking X-ray-derived redox changes in crystals of a methylamine dehydrogenase/amicyanin complex using single-crystal UV/Vis microspectrophotometry.

X-ray exposure during crystallographic data collection can result in unintended redox changes in proteins containing functionally important redox centers. In order to directly monitor X-ray-derived redox changes in trapped oxidative half-reaction intermediates of Paracoccus denitrificans methylamine dehydrogenase, a commercially available single-crystal UV/Vis microspectrophotometer was installed on-line at the BioCARS beamline 14-BM-C at the Advanced Photon Source, Argonne, USA. Monitoring the redox state of the intermediates during X-ray exposure permitted the creation of a general multi-crystal data collection strategy to generate true structures of each redox intermediate.

UV-induced fragmentation of Cajal bodies.

The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28gamma (proteasome activator subunit gamma). The presence of PA28gamma in coilin-containing complexes is increased by UV-C. Overexpression of PA28gamma, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference-mediated knockdown of PA28gamma attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28gamma as a novel regulator of CB integrity.

Radiation damage in macromolecular cryocrystallography.

X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.

Preparation of nano-sized particles from collagen II by a high-voltage electrostatic field system.

The pilot study describes a novel method for preparing nano-sized particles from collagen II using a high-voltage electrostatic field system. Observations from transmission electron microscopy showed that, in one of the cases, the nano-sized collagen II particles exhibited good sphericity, and the particles were in the range of 23.3+/-1.7 nm in diameter at the experimental setting of 3 kV cm(-1), for a 3 h treatment period and at 25 degrees C (with a collagen concentration of 0.2 mg ml(-1)). When the treatment temperature increased to 30 degrees C, the collagen II began to lose the tendency to form individually separated spherically shaped nano-particles. Moreover, a fibrous structure of collagen II was formed instead of a nano-particle shape at the temperature of 37 degrees C. This result is probably contributed to by an entropy-driven process that is termed fibrillogenesis, a larger force causing the collagen molecules to self-assemble and then form collagen fibrils. It is interesting to note that this is practically the first attempt to produce nano-particles directly from collagen II solution under the treatment of a high-voltage electrostatic field, together with a set of working parameters for the collagen concentration and low-temperature setting.

Radiation driven collapse of protein crystals.

During coherent X-ray diffraction measurements on crystals of ferritin at room temperature using monochromatic undulator radiation from the Advanced Photon Source, a sudden lattice contraction was observed following a characteristic latent period and ultimately leading to the collapse of the crystal. The progression of this collapse is analysed using a two-state Hendricks-Teller model. It reveals that 55% of the layers collapse by 1.6% before the crystal completely stops diffracting.

Electric birefringence study of an amyloid fibril system: the short end of the length distribution.

In this article, a system of amyloid fibrils, based on the protein beta-lactoglobulin, is studied by transient electric birefringence. Single pulses of an electric field were applied to the solution, and the initial rise and subsequent decay of birefringence analysed. The decay takes place on a range of relaxation times, and therefore contains information about the length distribution of fibrils in the system. The information can be extracted using theories of the electric polarisability of polyelectrolyte rods, since the fibrils are an example of these. Despite the long-standing complications of such theories, useful quantitative information about the system can still be obtained. Using the Fixman model of polyelectrolyte polarisability, we obtain a measurement of the short end of the length distribution which shows the fibril concentration as a function of length rising linearly from 0.02-2 microm. The short end of the length distribution was unobtainable in our previous study using rheo-optics (S.S. Rogers et al., Macromolecules 38, 2948 (2005)), but reasonable agreement between the two techniques shows they are complementary.

Processing of membrane protein crystal using ultraviolet laser irradiation.

We demonstrated the processing of a membrane protein crystal, using a pulsed UV laser soft ablation (PULSA) technique. Irradiation with deep-UV laser pulses at a wavelength of 193 nm successfully processed not only single crystals of the membrane transporter protein AcrB but also nylon loops and cryoprotectants at a cryogenic temperature. Nonprocessed parts of the crystals exhibited no signs of crack or denaturation after the laser exposure. The trimmed crystals were found to be of high resolution for X-ray diffraction data collection. The results described here indicate that PULSA processing is an effective tool for membrane protein crystals, as well as for soluble protein crystals.