Time-resolved Anisotropy Research Articles

Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein

The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of themutants. Using these data, we calculated dissociation constants of 1.9-3.3μM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.

Dynamics of Nuclear Receptor Helix-12 Switch of Transcription Activation by Modeling Time-Resolved Fluorescence Anisotropy Decays

Nuclear hormone receptors (NRs) are major targets for pharmaceutical development. Many experiments demonstrate that their C-terminal Helix (H12) is more flexible in the ligand-binding domains (LBDs) without ligand, this increased mobility being correlated with transcription repression and human diseases. Crystal structures have been obtained in which the H12 is extended, suggesting the possibility of large amplitude H12 motions in solution. However, these structures were interpreted as possible crystallographic artifacts, and thus the microscopic nature of H12 movements is not well known. To bridge the gap between experiments and molecular models and provide a definitive picture of H12 motions in solution, extensive molecular dynamics simulations of the peroxisome proliferator-activated receptor-γ LBD, in which the H12 was bound to a fluorescent probe, were performed. A direct comparison of the modeled anisotropy decays to time-resolved fluorescence anisotropy experiments was obtained. It is shown that the decay rates are dependent on the interactions of the probe with the surface of the protein, and display little correlation with the flexibility of the H12. Nevertheless, for the probe to interact with the surface of the LBD, the H12 must be folded over the body of the LBD. Therefore, the molecular mobility of the H12 should preserve the globularity of the LBD, so that ligand binding and dissociation occur by diffusion through the surface of a compact receptor. These results advance the comprehension of both ligand-bound and ligand-free receptor structures in solution, and also guide the interpretation of time-resolved anisotropy decays from a molecular perspective, particularly by the use of simulations.

Restricted State Selection in Fluorescent Protein Förster Resonance Energy Transfer

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Influence of surfactants on the photophysics of 4,4-diaminodiphenyl sulfone encapsulated in self-assembled nanocage of diblock copolymer

• Influence of cationic and non-ionic micelles drastically reduces the critical aggregation constant but not in the case of anionic micelle. • Addition of anionic micelle causes the probe to move toward the edge of the aggregation due to a possible disruption. • Further addition of polymer in polymer-anionic micelle gains rigidness with probe position unchanged. • Wobbling motion of the whole polymer-micelle assembly is fastest in non-ionic micelle with minimum semi-cone angle. This paper reports the influence of anionic, cationic and nonionic surfactant on the spherical nonionic polymeric micelles composed of block copolymers polyethylene- b -polyethylene glycol (PE- b -PEG) and the modulation of excited state emission characteristics of 4,4-diaminodiphenyl sulfone (Dapsone) in the mixed polymer-micelle environments. The critical micellar concentration (CMC) of diblock polymer was determined as ~0.12 mM from the variation in fluorescence intensity and solvatochromic shift of the probe with polymer concentration. Addition of surfactant in polymer solution of Dapsone causes a decrease in the value of critical aggregation concentration (CAC) to roughly one tenth the values of CMC of the individual surfactants (cationic or non-ionic) except in the case of anionic surfactant. The huge enhancement in fluorescence intensity and large blue shift of the emission maximum of the probe incorporated inside the nanocages formed by the diblock copolymer and cationic or nonionic micelle indicate an entirely different environment than that in neat water, polymer and micellar system separately. The steady state fluorescence anisotropy and dynamic light scattering (DLS) results on addition of micelle to the polymeric solution show two classes of events; (1) addition of anionic micelle causes lesser rotational restriction and (2) addition of cationic and non-ionic micelles cause to form more rigid environment to the probe. The rotational motion of the probe analyzed in terms of “wobbling–in-cone” model with the fluorescence anisotropy decay data shows that the wobbling is fastest in non-ionic micelle with minimum semi-cone angle. The increased rotational time and the value order parameter obtained from the time-resolved anisotropy decay confirm a very restricted environment experienced by the probe inside the self assembly system. The translational and the wobbling motions are also restricted inside these aggregations.

Photophysics of Delocalized Excitons in Carbazole Dendrimers

The photophysical properties in solution of three generations of carbazole-based dendrons and dendrimers with fluorenyl surface groups were studied using steady-state, time-resolved femtosecond transient absorption and anisotropy, and coherent two-dimensional ultraviolet spectroscopy. It was found that increasing the generation caused a switch in the nature of the emissive state between the first-generation compounds and the second- and third-generation dendrimers. Time-resolved anisotropy measurements revealed low initial anisotropies that decreased with increasing dendrimer generation consistent with increasing intradendrimer interchromophore coupling. Two-dimensional UV spectroscopy showed that the signal from the second- and third-generation dendrimers is the product of multiple chromophores interacting. The maximum number of interacting chromophores is reached by the second generation.

Understanding the Spectroscopic Properties and Aggregation Process of a New Emitting Boron Dipyrromethene (BODIPY)

Aggregation of organic dyes often has consequences on their spectroscopic properties in materials. Here, we study a new sterically hindered boron-dipyrromethene (BODIPY), with adamantyl moieties grafted for the first time on the BODIPY core. Its aggregation behavior was investigated in poly(methyl methacrylate) (PMMA) and on drop-casted films by monitoring absorption, fluorescence emission, relative quantum yield (ΦFluo,Rel), lifetime and time-resolved anisotropy. Aggregates only appear from 0.067 mol·L–1. A multicomponent analysis demonstrated that the aggregation process can be described by three distinguishable components which correspond to a monomer species (M) and J and H aggregates. The results also indicated a concentration frontier: when the dye concentration increased up to 0.29 mol·L–1, the concentration of M decreased in favor of the aggregates. ΦFluo,Rel is yet only divided by 5 compared to the dye in solution. Above 0.29 mol·L–1, an equilibrium between M and the J aggregates is established, showing meanwhile a steady ΦFluo,Rel. The J aggregates are found to be dimers, whereas the aggregation number is varying for the H aggregates. Analysis of fluorescence and anisotropy decays showed that the excitation energy was transferred from M to the J dimers, and very probably trapped by H aggregates.

The Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

We have used time-resolved phosphorescence anisotropy to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin concentrations but substantially higher apparent actin affinity, resulting in a higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy cooperatively and to a similar extent, indicating a similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of a saturating level of ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin.

Conformational dynamics and excitation wavelength dependent photoluminescence of decameric organic nanoparticles

We report a new type of fluorescent organic nanoparticles (16-EDFONP) which are composed of ten bis(3,4,5-tris(16-alkyloxy) monobenzoylglyceride) bisphenol A molecules. The nanoparticles are only found in high concentration solution and exhibit the excitation-dependent fluorescence (EDF). The 16-EDFONP shows clear spectral relaxation on the nanosecond time scale. We have observed similar spectral shifts in cyclohexane, and thus attribute the time-dependent Stokes shift to the emission from multiple conformations of 16-EDFONP. With the employment of steady state and time-resolved fluorescence anisotropy measurements, the hydrodynamic radius of 16-EDFONP is estimated to be 3.13 nm, which is consistent with the size measured using the dynamic light scattering and high-resolution transmission electron microscopy techniques. The time-resolved anisotropy reveals the change in fundamental anisotropy upon different excitation wavelengths, arising from the structural heterogeneity of hydrogen-bonded monoacylglycerol clusters of the 16-EDFONP. Our findings indicate that incomplete spectral relaxation and the size distribution of nanoparticles are not the source of the observed EDF. The EDF comes from the selective excitation of the 16-EDFONP with different monoacylglycerol hydrogen-bond conformations.

Homoconjugation in diporphyrins: excitonic behaviors in singly and doubly linked Zn(ii)porphyrin dimers

We have comparatively investigated excitonic features between singly and doubly alkyl bridged Zn(II)porphyrin dimers (SLZn and DLZn, respectively) with Zn(II)tetraphenylporphyrin (ZnTPP) as a reference by using various time-resolved anisotropy measurements. Time-resolved fluorescence anisotropy decay of the B-state of ZnTPP in toluene showed ultrafast dephasing times of 83 and 185 fs with an initial anisotropy value (r0) of ∼0.7 being consistent with the theoretically proposed one. On the other hand, the Q-state of ZnTPP exhibited a depolarization time of 120 fs with a smaller r0 of 0.25 than that of the B-state because of probing photo-induced absorption in transient absorption anisotropy measurements. Spectroscopic features in absorption, fluorescence, and excitation anisotropy spectra of SLZn are similar to those of ZnTPP indicating a relatively weak exciton coupling in the Q-state. As a result, the observed depolarization time with a time-constant of 1.4 ps can be explained by a Forster-type incoherent energy transfer process between two constituent subunits. When compared to ZnTPP and SLZn however, DLZn exhibited very different optical characteristics such as more red-shifted absorption and emission spectra, three times larger fluorescence excitation anisotropy values, and ultrafast sign inversion in transient absorption anisotropy. According to these results, it can be inferred that the overall π-electron densities of the electronic excited states in DLZn are fully delocalized in a whole dimer and that DLZn can be regarded as a coherently coupled single quantum system not as separate individual chromophores. Frontier π-molecular orbital structures as well as electron density difference maps between occupied and unoccupied molecular orbitals involved in the electron promotions for underlying excited states clearly represent weakly and strongly exciton-coupled natures of SLZn and DLZn, respectively. Accordingly, the extended conjugation of π-electrons over the whole DLZn molecule demonstrates an example for homoconjugation between two aromatic porphyrins.

Clustering of Ras on Membrane Surfaces Independent of Lipid Anchor Effects

Ras is a lipid-anchored small molecule GTPase and an important signaling node in mammalian cells. The dynamics of subcellular localization are a crucial dimension of Ras signaling, observed as lateral partitioning and hierarchies of lipid and protein interactions. H-Ras clustering in reconstituted bilayer systems was studied using a combination of photon counting histogram (PCH) spectroscopy, time-resolved anisotropy, and single molecule (SM) tracking. Lipid anchor effects were specifically excluded by tethering the hypervariable region (HVR) of H-Ras directly to membrane-miscible lipid tails in these bilayers. Translational and rotational diffusion were characterized across a broad range of surface densities. PCH revealed the H-Ras degree of clustering, which was confirmed by SM tracking. Using these complementary spectroscopic techniques, we calculated the dissociation constants of H-Ras in its nucleotide-dependent active and inactive states. Strikingly, we identified a point mutation that alters H-Ras clustering. We will present insight into the lipid membrane's role in this Ras oligomerization interaction, and discuss the implications for the regulation of up- and downstream effectors.

Evidence of concerted inversion for the photon-induced molecular switching of azobenzene using rotation-free azobenzene derivatives

The excited state relaxation of two types of azobenzene derivative excited at 350 nm in the short-wavelength (S2(π, π*)) band was studied by transient absorption spectroscopy. We compared the relaxation dynamics and the time-resolved anisotropic relaxation properties of these two molecules in liquid solution and in bulk film. The average relaxation life-time for Azo-2 in bulk film (13.2 ps) is much slower than that of Azo-2 (4.06 ps) and Azo-1(1.31 ps) in liquid solution. The time-resolved polarization anisotropy spectroscopy measurements of these samples also showed significant differences. The value of anisotropy for Azo-2 remained almost constant at about 0.2 in bulk film but decayed slowly from 0.3 in liquid solution. The value for Azo-1 in liquid solution decayed very fast from 0.4 to 0.2 within 6 ps. Our results gave new evidence for the existence of a converted inversion channel. We also concluded that the concerted inversion process and the conventional rotation process govern the relaxation for the photon-induced switches of azobenzene and its derivatives under short-wavelength band excitation.

Characterizing the Interactions of Lysophospholipids with Lipid Membranes

This study aims to characterize and compare thermodynamic interactions of the lysophospholipid - C12 - lysophosphocholine (lysoPC) and its synthetic analog, n-dodecylphosphocholine (DPC) - with lipid membranes. As a biomolecule possessing detergent-like properties, lysoPC is involved in many biological processes and DPC has been used widely in NMR studies of membrane proteins. We investigate the lipid-detergent systems by determining partition coefficient, mole ratios of bound detergent to lipid at membrane saturation and solubilization boundaries, and the mechanism of membrane disordering and pore formation. Isothermal Titration Calorimetry (ITC) is used for assays such as demicellization, uptake-and-release and solubilization-and-reconstitution. Time-resolved DPH anisotropy and lifetime-based leakage assays are used to study membrane structural changes upon detergent incorporation in liposomes. Both lysoPC and DPC equilibrate with membranes very slowly. We hypothesize that the free energy penalty due to asymmetric membrane insertion limits the membrane uptake of lysoPC and DPC. This would be at variance to other detergents that induce membrane failure above a threshold asymmetry. Results are important for understanding mechanisms for membrane protein isolation and the interactions of amphiphilic biological compounds with lipid membranes.

Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

Muscle contraction results from cyclic interaction of myosin and actin, driven by ATP hydrolysis, where the actomyosin complex undergoes transitions between weakly and strongly bound structural states. Rabbit skeletal muscle myosin has two isoforms of the essential light chain (ELC), designated A1 and A2. These isoforms have significant effects on myosin's interaction with actin, resulting in higher catalytic efficiency for A1 but higher in vitro motility and muscle shortening velocity for A2. In order to understand the structural basis of these differences, we have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of ELC isoforms on the microsecond dynamics of actin during interaction with skeletal muscle myosin in the absence and presence of saturating ATP. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes containing A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy similarly, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (rigor) state of actomyosin. In contrast, in the presence of saturating ATP, the final anisotropy of actin was substantially higher during interaction with S1A1 than with S1A2. Thus, S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin, suggesting greater population of the strong-binding state of actomyosin. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, and thus modulates the contractile function of actomyosin.

The Effect of Small Regulatory RNA on Globular Protein Aggregation

Small regulatory RNA segments, such as microRNA (miRNA) or silencing RNA (siRNA) binding to proteins could effect the association mechanisms of protein aggregation. Extended protein aggregation, known as fibrillization, has links to degenerative diseases like Alzheimers or prion disease. The formation of fibrils is thought to be a non-specific property of proteins, and can be demonstrated with well-known model globular proteins like serum albumin, lysozyme, beta-lactoglobulin and the like. Much research has been devoted to this field, but with the recent discovery of micro/silencing RNA, small regulatory RNA generally less than 70 base pairs, the question arises on the effect of these nucleic acids on the aggregation process behind fibril formation. Therefore, this study attempted for the first time to probe two effects in the fibrillization process: first, the binding affinity of the selected microRNA MIR106A to the model proteins lysozyme and bovine serum albumin in fibril forming conditions, and second, the long-term effect of the protein-nucleic acid complex on the fibril formation process. Fluorescence spectroscopy, to include time-resolved anisotropy decay of fluorescein or dansyl-labeled complexes, and sizing techniques like atomic force microscopy to track aggregation patterns were incorporated.

Magnesium-Dependence and Active Site Dynamics in Myosin V and Myosin II

We examined the magnesium-dependence of heavy meromyosin V (MV HMM) and two class II muscle myosins, skeletal muscle myosin (SK HMM) and smooth muscle myosin (SM HMM), in the in vitro motility and solution actin-activated ATPase assays. We find that all three myosins are inhibited in a Mg2+-dependent manner (0.5-10 mM MgCl2) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. Interestingly, MV was more steeply dependent on magnesium than both SK and SM. In addition, actin filament breaking in the motility assay was two-fold faster with MV HMM at high (10 mM) compared to low (1 mM) MgCl2 conditions. To begin to understand how the active site of myosin V is different from myosin II we examined the time resolved fluorescence from dmantADP bound to myosin V 1IQ (MV 1IQ) or Dicty myosin II S1 (Dicty S1) in the presence of 1 mM MgCl2. We found that dmantADP bound to acto-MV 1IQ contained two lifetime components (8 and 3 ns). Time-resolved anisotropy studies of the two lifetime components in myosin V reveal that the long lifetime component was more immobilized (correlation time = 12 ns) while the short lifetime component was highly dynamic (correlation time = 0.4 ns). Interestingly, Dicty S1 contained a single lifetime component (8 ns). The two conformations of the myosin V active site may allow Mg2+ to more efficiently bind and reduce key steps in the ATPase cycle such as ADP release. Overall, our results suggest that differences in the structural dynamics of the active site of myosins may play a role in their dependence on free Mg2+, which could explain why Mg2+ differentially alters the motile and force generating properties of myosins.

Fluorescence Polarization and Fluctuation Analysis Reveals CAMKIIα Catalytic Domain Pair Extension Triggered by Ca2+/Calmodulin

The autoinhibited calcium/calmodulin-dependent protein kinases-II (CaMKII) assembles to form a holoenzyme structure with multiple catalytic domain pairs radiating out from a central core composed of oligomerization domains. Activation of CaMKII upon binding calcium2+/calmodulin (CaM) is known to initiate a biochemical cascade resulting in CaMKII translocation to synaptic spines and the induction of long-term potentiation. Little is known about the structural changes in the intact CaMKII holoenzyme responsible for this activation. Fluorescence polarization and fluctuation analysis (FPFA) microscopy, a method that combines time-resolved anisotropy and fluorescence correlation spectroscopy (FCS), was used to monitor Venus-CaMKIIα (where a Venus fluorescent-protein is tagged onto the N-terminus of CaMKIIα) before and after activation with CaM. FPFA simultaneously monitors brightness (a measure of the number of subunits in the holoenzyme), homo-FRET (a catalytic domain proximity detector) and diffusion time (a parameter sensitive to the mass and hydrodynamic volume of the holoenzyme). Together these measurements can potentially detect an array of different covert structural changes in the CaMKII holoenzyme triggered by CaM. In homogenates prepared from HEK cells expressing Venus-CaMKIIα, the addition of CaM triggered T286 autophosphorylation, an indicator of kinase activation. FPFA revealed a large increase in the holoenzyme diffusion time. Because changes in holoenzyme subunit stoichiometry were not observed, the change in diffusion time most likely indicates a large change in hydrodynamic volume, such as catalytic domain pair extension. This change was not observed in Venus-CaMKIIα-TT305/6DD, a mutant that cannot bind calmodulin. Holoenzyme activation by CaM did trigger a small, dose-dependent biphasic change in anisotropy (homo-FRET between Venus-tagged catalytic domains), but its magnitude was too small to be interpreted as catalytic domain un-pairing. Interpretation of these experiments will be discussed.

Advancements in laboratory diagnostics: an invaluable tool for assessing quality of blood transfusions.

The interesting article by Willy A. Flegel that recently appeared in this journal provides valuable insights into an ongoing debate on the use of fresher material for blood transfusions1. Regardless of important considerations about the delicate balance between selection of units and blood supply, an innovative approach for defining the quality of transfused components is increasingly supported by recent advancements in laboratory diagnostics, which would enable to determine whether leucocytes, platelets and red blood cells are suitable for transfusion regardless of the ageing of the stored material. Several examples can be given, whereby routine, automatic flow haemocytometry combined with additional technical tools provides valuable information. As regards platelets, the loss of function during storage is mirrored by disc-to-sphere transformation, extension of pseudopodia, alteration of the canalicular system and granules, up to loss of storage granules2. Some haemocytometers already provide a variety of activation-related information about these blood elements. For example, in Advia 2120 (Siemens Diagnostics, Tarrytown, New York, United States of America), the intensity of light scattered by platelets is assessed at two different angles (i.e., 2 to 3° and 5 to 15°). Mean platelet volume and mean platelet component are then calculated and from these paired measures. The latter parameter, in particular, has been shown to be a reliable index of platelet degranulation, since it is directly related to CD62 expression, which is highly indicative of platelet activation and ageing3. The same instrument, along with others such as the PENTRA 60C (Horiba-ABX Diagnostics, Montpellier, France), provide specific flags or alerts in the presence of degranulation of polymorphonuclear leucocytes4, which is a hallmark of functional impairment5. More specifically, the Advia 2120 also enables reliable quantification of the number of degranulated neutrophils in specific clusters of the peroxidase scattergram based on cell volume and intracellular myeloperoxidase concentration. Additional morphological changes that characterise gradual deterioration of leucocytes in stored blood include a more homogeneous staining of nuclei, the separation of the nuclear lobes or irregular lobulation and vacuolisation, which can be reliably identified in the scattergram of modern haemocytometers such as those of the Sysmex XE series, in which the intensity of side scatter is dependent on the complexity of nuclear lobulation and granules within the cytoplasm6. It is also noteworthy that several changes occur to red blood cells during medium- and long-term storage in blood bags, including reduced deformability, altered adhesiveness and aggregability, and decreased 2,3-diphosphoglycerate and ATP content, to the point of frank injury and spurious haemolysis7. Specific macromolecular remodelling, along with lipid peroxidation and/or oxidative damage to the membrane, are mainly responsible for the onset of irreversible morphological abnormalities and loss of function. Most of these physicochemical changes, usually referred to as the “storage lesion”, can be identified by time-consuming or expensive analytical techniques such as flow cytometry, time-resolved phosphorescence anisotropy and microscopy, which are unsuitable for rapid examination of a large number of blood bags8. Nevertheless, the presence of red blood cell crenation or fragmentation -and finally haemolysis- can now be reliably and rapidly identified by specific erythrocyte indices on automated flow haemocytometers, such as the “immature platelet fraction” on XE-2100 (Sysmex, Kobe, Japan), or “RBC ghosts” on Advia 21209. Another interesting approach is ThromboLUX™ (LightIntegra Technology Inc., Vancouver, British Columbia, Canada), which uses the principle of dynamic light scattering -also known as photon correlation spectroscopy or quasi-elastic light scattering- to identify and quantify the particles that may be present in a platelet concentrate, as well as showing how they respond to temperature stress. The instrument finally generates a quality score on a scale ranging from 0 to 40, with platelet concentrates scoring from 13 to 40 containing mostly discoid and functional platelets. Recent data show that the information from ThromboLUX™ correlates adequately with that from flow cytometry and microscopy and may, therefore, be suitable for quality assessment of platelet concentrate samples10. Several technical advances have occurred in laboratory diagnostics over the past decades. These have not only improved the global quality of testing, but have also enabled reliable application to transfusion medicine for the assessment of the quality of transfused components, as attested by some of the examples previously provided. The modern, fully-automated haemocytometers -which are characterised by high accuracy and precision, notable throughput and full integration with information systems- can also provide a kaleidoscope of cellular indices. Most of these novel parameters are still partially unexploited, but it is conceivable that they may be reliably used to detect artefactual changes in leucocytes, platelets and red blood cells, which may be suggestive of less functional, defective or even apoptotic cells, and finally establish as to whether cellular elements within blood bags are still viable for transfusion. Future studies should be focused on the evaluation of the usefulness of these parameters in the setting of transfusion medicine.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Unusual Optical Resolution of All Four Tryptophan Residues in MPT63 Protein by Phosphorescence Spectroscopy: Assignment and Significance

MPT63, a secreted protein of unknown function that is specific to Mycobacterium tuberculosis and a potential drug target, contains four Tryptophan (Trp/W) residues located at positions 26, 48, 82, and 129 in the amino acid sequence. All of the four Trp residues have been optically resolved by simple inexpensive phosphorescence spectroscopy at 77 K. The protein architecture provides a delicate micro-environment and location of Trp residues giving rise to four different (0,0) bands in the phosphorescence spectra. Calculation of intra Trp energy transfer (ET) efficiency, accessible surface area (ASA) of Trp residues, and environment of Trp in the wild-type (WT) and the mutant W26F [where, Trp 26 is replaced by phenyl alanine (Phe/F)] reveal: E(T1) (W82) > E(T1) (W48) > E(T1) (W129) > E(T1) (W26), where E(T1) is the lowest (π-π*) triplet state energy of Trp. The (0,0) band observed at 421.6 nm assigned for Trp 26 is found to be the longest wavelength (0,0) band so far reported in the literature. Fluorescence in WT and W26F is dominated by buried or partially exposed Trp residues indicated by time-resolved spectra. Circular Dichroism (CD) studies and the time-resolved anisotropy measurement confirm the unaltered secondary and tertiary structure of the mutant compared to that of the WT. Excitation energy dependent phosphorescence spectra suggest that the intensity of the different (0,0) bands could be tuned and Tyrosine (Tyr/Y) residue is silent in emission. Optical resolution of all the Trp residues will help understand the role of each Trp residue in the folding/unfolding mechanism and in the interaction with other systems.

Highly luminescent Ru(ii) metallopolymers: photonic and redox properties in solution and as thin films

Highly luminescent metallopolymers have been prepared from condensation reactions between the primary alkyl amines contained within a poly(styrene-p-(aminomethyl)styrene) polymer backbone and carboxylic acid groups on [Ru(bpy)(2)(CAIP)](ClO(4))(2), where CAIP is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. The photophysical properties of these metallopolymers including emission and absorption spectroscopy, luminescent lifetimes and time-resolved emission anisotropy show strong solvent and pH dependence. These properties are rather insensitive to the loading of the metal centres on the polymer backbone suggesting weak electronic interactions between adjacent centres and the polymer backbone. The polymers form continuous films when drop-cast at an indium tin oxide interface and retain their strong luminescence. This processability and intense luminescence make them potentially useful for sensing and display applications.