Models of the actin monomer and filament from fluorescence resonance-energy transfer.

We have developed algorithms for combining fluorescence resonance-energy transfer (FRET) efficiency measurements into structural models which predict the relative positions of the chemical groups used in FRET. We used these algorithms to construct models of the actin monomer and filament derived solely from FRET measurements based on seven distinct loci. We found a mirror-image pair of monomer models which best fit the FRET data. One of these models agrees well with the atomic-resolution crystal structure recently published by Kabsch et al. in Heidelberg [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37-44]. The root-mean-square deviation between this FRET model and the crystal structure was about 0.9 nm. Other macromolecular models assembled from FRET measurements are likely to have a similar resolution. The largest discrepancy was for the Cys10 locus which deviated 1.44 nm from the crystal position. We discuss the limitations of the FRET method that may have contributed to this discrepancy, and conclude that the Cys10 FRET data have probably located Cys10 incorrectly in the FRET monomer model. Using the FRET monomer models, we found three orientations in the filament which best fit the intermonomer FRET data. These orientations differ substantially from the atomic-resolution filament model proposed by the Heidelberg group [Holmes, K., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49], largely because of the discrepancies in the Cys10 data. These data should probably be excluded from the analysis; however, this would leave too few measurements to assemble a filament model. In the near future, we hope to obtain additional FRET measurements to other actin loci so that the filament modelling can be done without the Cys10 data.     

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.     

Mechanistic insight from chaos: how RecA mediates DNA strand exchange

Cleverly designed single-molecule FRET experiments reported in this issue of Structure by Ragunathan et?al. coax RecA to reveal some of its secrets. Observing individual events identifies intermediate steps and provides clues for how to drive strand exchange forward.     

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor''s fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor''s emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005). Consequently, conventional imaging of GFP and its closest spectral variants (like cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP]) is most likely to be problematic in roots, whereas red-shifted intrinsic fluorescent proteins (including monomeric red fluorescent protein and recently identified spectral variants like mStrawberry and mCherry) may be hard to discriminate in chloroplast-containing aerial tissues (Chapman et al., 2005). The problems get further compounded in FRET assays because the autofluorescence arising from phenols, lignin, and chlorophyll can limit the choice of fluorophores suitable for in planta FRET assays.CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006; Dixit et al., 2006). However, in photophysical terms, this pair is less than ideal for FRET imaging. Both have broad excitation and emission spectra with a small Stokes shift (Chapman et al., 2005). Second, QY of CFP (QY = 0.4) is relatively lower than that of YFP (QY = 0.61), and thus a significantly higher (and rather cell damaging) amount of excitation energy is needed to induce FRET (Dixit et al., 2006). Additionally, CFP displays multiexponential lifetimes with a shorter (1.3 ns) and a longer (2.6 ns) component (Becker et al., 2006). Although the deviation from the single-component decay is reasonably small (Tramier et al., 2002; Becker et al., 2006), the shorter CFP lifetime component can erroneously be interpreted as being the result of lifetime reduction due to energy transfer. At the same time, weak or transient protein associations may get masked and thus remain undetected. Whereas the parental wild-type GFP is extremely photostable and shows a monoexponential decay pattern (excited-state lifetime 3.16 ± 0.03 ns; Striker et al., 1999; Volkmer et al., 2000; Shaner et al., 2005), its close spectral overlap with YFP makes it unsuitable as a donor in GFP-YFP FRET experiments. Likewise, wild-type or enhanced GFP (or YFP) as a donor to red-shifted monomers as acceptors is suboptimal because the 488-nm (or 514-nm) laser line commonly used to excite GFP (or YFP) cross excites most of the red monomers (e.g. mOrange, mStrawberry) because of their broad excitation spectra (Zapata-Hommer and Griesbeck, 2003; Shaner et al., 2004).Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003). The T203I mutation in TSapphire (and original Sapphire as well) abolishes the 475-nm excitation peak found in the wild-type GFP (Tsien, 1998). TSapphire is efficiently excited below 410 nm, which makes it ideal for studying plant protein dynamics and interactions because, at this wavelength, there is negligible excitation of the autofluorescing chlorophyll pigments. Furthermore, TSapphire also represents a good donor to red monomer acceptors that are negligibly excited at this wavelength (Shaner et al., 2004). Using a purified Zn²⁺ sensor with TSapphire and mOrange as a donor-acceptor pair, Shaner and colleagues demonstrated the ratiometric intramolecular FRET between the two fluorophores in vitro (Shaner et al., 2004). The sensor yielded a 6-fold ratiometric increase (562/514-nm mOrange/TSapphire emission ratio) upon Zn²⁺ binding.However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999; Volkmer et al., 2000). Furthermore, we demonstrate intramolecular FRET-FLIM between chimerically linked TSapphire and mOrange (with a 16-amino acid linker in between). When fused to genetically known interacting proteins and expressed in intact living cells, the donor and the acceptor fluorophores show energy transfer in different subcellular compartments indicative of intermolecular protein-protein interactions. These results validate the versatility of the proposed in vivo FRET-FLIM assay based on the donor TSapphire and the acceptor mOrange, which turns out to work with both soluble and membrane proteins.     

Real‐time detection of cellular death receptor‐4 activation by fluorescence resonance energy transfer

Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL–DR4 in live cells in real‐time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4‐CFP and DR4‐YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4‐CFP and DR4‐YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4‐CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4‐CFP/YFP reporter cells exhibited a colocalized expression of DR4‐CFP and DR4‐YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi‐fluorescence microscopy for FRET analysis. The real‐time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time‐lapse fluorescence microscopy. Therefore, DR4‐CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti‐cancer drug screening to identify compounds that are capable of activating TRAIL pathways. Biotechnol. Bioeng. 2013; 110: 1396–1404. © 2012 Wiley Periodicals, Inc.     

New turf for CFP/YFP FRET imaging of membrane signaling molecules

TIRF microscopy can be used in conjunction with CFP/YFP FRET to detect movements of the cytoplasmic tails of GIRK channels (Riven et al., this issue of Neuron). This innovative combination of techniques allows molecular resolution of small motions underlying ion channel activation by G proteins and will likely find widespread use for study of membrane-associated molecules.     

Non-3D domain swapped crystal structure of truncated zebrafish alphaA crystallin

In previous work on truncated alpha crystallins (Laganowsky et al., Protein Sci 2010; 19:1031–1043), we determined crystal structures of the alpha crystallin core, a seven beta‐stranded immunoglobulin‐like domain, with its conserved C‐terminal extension. These extensions swap into neighboring cores forming oligomeric assemblies. The extension is palindromic in sequence, binding in either of two directions. Here, we report the crystal structure of a truncated alphaA crystallin (AAC) from zebrafish (Danio rerio) revealing C‐terminal extensions in a non three‐dimensional (3D) domain swapped, “closed” state. The extension is quasi‐palindromic, bound within its own zebrafish core domain, lying in the opposite direction to that of bovine AAC, which is bound within an adjacent core domain (Laganowsky et al., Protein Sci 2010; 19:1031–1043). Our findings establish that the C‐terminal extension of alpha crystallin proteins can be either 3D domain swapped or non‐3D domain swapped. This duality provides another molecular mechanism for alpha crystallin proteins to maintain the polydispersity that is crucial for eye lens transparency.     

133 Designed DNA crystals with a triple-helix veneer

DNA has been used as a tool for the self-assembly of nano-sized objects and arrays in two and three-dimensions. Triplex-forming oligonucleotides (TFOs) can be exploited to recognize and introduce functionality at precise duplex regions within these DNA nanostructures (Rusling et al., 2012). Here we have examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry, consisting of three helices directed along three linearly independent directions (Liu et al., 2004). The triangles form a three-dimensional crystalline lattice stabilized via sticky-end cohesion (Zheng et al., 2009). The TFO 5′-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine–oligopyrimidine binding site. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET analysis. Non-denaturing gel analysis of annealed DNA motifs showed a band with slower mobility only in the presence of TFO and only when the DNA motif contained the triplex binding site. Experiments were undertaken at pH 5.0, since the formation of a triplex with cytidine-containing TFOs requires slightly acidic conditions (pH<?6.0). TFOs with modified C-analogs and T-analogs having a higher pK _a worked at a more neutral pH, also evidenced by EMSA. UV melting studies revealed that the melting point of the 3-turn triangle was 64?°C and the TFO binding increased the melting point to 80?°C. FRET analysis was done by labeling the triangle with fluorescein and the TFO with a cyanine dye (Cy5). The FRET melting curve revealed that a signal was observed only when the TFO was bound to the DNA motif and the results were consistent with UV melting studies. These results indicate that a TFO can be specifically targeted to the tensegrity triangle motif.     

Journal of Cellular Biochemistry: Volume 112, Number 10, October, 2011

Cover: The cover shows a schematic illustrating D‐loop extension in the T4 recombination‐dependent replication system that is coupled to assembly of the replicative DNA helicase. See Prospect in this issue by Maher et al, pages 2672–2682.     

A Mutation in the LPAT1 Gene Suppresses the Sensitivity of fab1 Plants to Low Temperature

Various fluorophore-based microscopic methods, comprising Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.Many biological processes rely on the dynamic assembly and disassembly of multicomponent protein complexes. Experimental methods based on genetically encoded fluorophores are widely used to study the subcellular localization and binary protein-protein interactions in living cells. The subcellular localization of proteins can be visualized by translationally fusing them to fluorophores, which are now available across a wide spectral range (Shaner et al., 2005). Fluorescence microscopy allows imaging and tracing of these proteins in real time in living cells. However, the resolution of a conventional epifluorescence microscope or a confocal laser scanning microscope is insufficient to resolve distances smaller than approximately 200 nm between biological macromolecules and to demonstrate their vicinity at the molecular scale (Bhat et al., 2006; Held et al., 2008).Two fluorophore-based methods are commonly employed to study protein-protein interactions on the stage of a light microscope and overcome its limitations: bimolecular fluorescence complementation (BiFC; Hu et al., 2002; Bracha-Drori et al., 2004; Walter et al., 2004) and Förster resonance energy transfer (FRET; Hink et al., 2002; Chen et al., 2003; Russinova et al., 2004). The BiFC approach is based on the restoration of an intact fluorophore from its nonfluorescent N- and C-terminal domains. When the two complementing fragments are fused to potentially interacting proteins, close proximity of the proteins can bring the N- and C-terminal fluorophore domains into a favorable position and orientation and thereby facilitate their association into a functional fluorophore (Hu et al., 2002; Kerppola, 2008). Since only standard fluorescence microscopic equipment is required for this technique, it is a popular method to analyze protein-protein interactions (Weinthal and Tzfira, 2009).An alternative procedure exploits a physical phenomenon referred to as FRET to obtain information about the molecular vicinity of fluorophore-labeled proteins. FRET occurs when a donor fluorophore is brought into close proximity (less than 10 nm) of a suitable acceptor fluorophore. In this case, the donor can transmit its excitation energy to the acceptor. The extent of FRET depends on various parameters, such as the distance between donor and acceptor, their spectral properties, the relative orientation of the donor and acceptor transition dipoles, and the refractive index of the medium (for a more detailed introduction to the theory of FRET, see “Materials and Methods”; Clegg, 1996; Periasamy and Day, 2005; Biskup et al., 2007). The extent of FRET can be estimated by several means, such as by assessing the sensitized emission (Shah et al., 2001, 2002), by determining the donor fluorescence before and after acceptor photobleaching (APB; Bhat et al., 2005), or by measuring the fluorescence lifetime of the donor (Russinova et al., 2004; Bhat et al., 2005; Tonaco et al., 2006; Shen et al., 2007; Osterrieder et al., 2009). Depending on the method, FRET measurements require more or less sophisticated microscopic equipment.Many biological processes involve protein complexes composed of more than two proteins, which are difficult to study with the experimental approaches outlined above. For example, biologically active N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complexes are composed of three distinct protein partners. SNAREs, together with accessory proteins and cytosolic calcium, catalyze membrane fusion events in eukaryotic cells. A binary or ternary t-SNARE complex composed of a Qa-SNARE, also called syntaxin, and Qb- and Qc-SNARE domains present in either one protein (synaptosome-associated protein [SNAP-25]) or as two separate units is located at the target membrane. This t-SNARE complex interacts with an R-SNARE (vesicle-associated membrane protein [VAMP]) present on the vesicle, and the formation of the fusogenic SNARE complex brings the opposing membranes into close proximity (Jahn and Scheller, 2006; Leabu, 2006). Besides their role in cellular homoeostasis, SNAREs play a major role in various plant biological processes, such as cytokinesis, gravitropism, and defense against pathogens (Lipka et al., 2007).The orthologous Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare) syntaxins, AtPEN1 and HvROR2, respectively, are essential in restricting the invasion of nonadapted powdery mildew species or in broad-spectrum powdery mildew resistance conferred by loss-of-function mlo mutants, respectively (Collins et al., 2003; Assaad et al., 2004). Both syntaxins form a binary t-SNARE complex with the orthologous SNAP-25-like proteins AtSNAP33 and HvSNAP34 (Collins et al., 2003; Kwon et al., 2008), which in turn interact with the R-SNAREs AtVAMP721/AtVAMP722 and HvVAMP721, respectively, by adopting an authentic ternary SNARE complex (Kwon et al., 2008). In addition to a range of other polypeptides, the mentioned Arabidopsis and barley SNARE partners focally accumulate below the site of attempted fungal penetration (Assaad et al., 2004; Bhat et al., 2005; Kwon et al., 2008), which probably precedes their extracellular deposition in the paramural space (Meyer et al., 2009).In this study, we combined BiFC-FRET measurements based on cerulean fluorescent protein (CrFP; Rizzo et al., 2004) as a donor and reconstituted yellow fluorescent protein (YFP; Walter et al., 2004; Schütze et al., 2009) as an acceptor to study the formation of a ternary protein assembly. We concentrated on the well-characterized SNARE complexes described above that play a decisive role in antifungal defense. FRET was detected by measuring the fluorescence lifetime of the donor in each pixel of the sample in a fluorescence lifetime imaging (FLIM) setup. Occurrence of FRET was corroborated by APB experiments. Our data demonstrate the technical feasibility of combined BiFC-FRET-FLIM and BiFC-FRET-APB assays to study ternary protein complexes in living plant cells.     

Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12

Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type resonance energy transfer (FRET). Two single-cysteine mutants (K132C and N244C) were labeled with either Alexa-532 (donor) or Alexa-647 (acceptor). The labels were positioned at the sites thought to be on the cis side of the known transmembrane regions [Ladokhin, A. S., et al. (2002) Biochemistry 41, 13617]. If the pore were comprised of an annexin oligomer, efficient energy transfer should be observed. Fluorescence excitation spectra of several mixtures of donor- and acceptor-labeled ANX were recorded under various conditions. Spectroscopic hallmarks of oligomerization-related FRET were established by following a well-documented transition of ANX from the soluble monomer to surface trimer upon addition of calcium at neutral pH. These hallmarks, however, were not detected for the membrane-inserted form of ANX at pH 4.5, suggesting that the transbilayer form is a monomer. This implies that the pore is formed by several transmembrane regions of the same ANX molecule. FRET and other fluorescence experiments demonstrate that the transitions between the surface trimer and membrane-inserted monomer are reversible. This reversibility, in combination with the absence of oligomerization in the water-soluble and inserted state, makes ANX a good experimental model for thermodynamic studies of folding and stability of membrane proteins.     

160 A dynamic model for the linker histone

Linker histones play an important role in the packing of chromatin. This family of proteins generally consists of a short, unstructured N-terminal domain, a central globular domain, and a C-terminal domain (CTD). The CTD, which makes up roughly half of the protein, is intrinsically disordered in solution but adopts a specific fold upon interaction with DNA (Fang et al., 2012). While the globular domain structure is well characterized, the structure of the CTD remains unknown. Sequence alignment alone does not reveal any significant homologs for this region of the protein. Construction of a model thus requires additional information. For example, the atomic model for the rat histone H1d CTD, proposed over a decade ago, used novel bioinformatics tools and biochemical data (Bharath et al., 2002). New fluorescence resonance energy transfer (FRET) studies of the folding of the CTD in the presence of linear DNA, single nucleosomes, and oligonucleosomal arrays (Caterino et al., 2011; Fang et al., 2012) have stimulated our interest in constructing a dynamic model of the protein. We have obtained preliminary information about the structure and dynamics of the linker histone CTD through ab initio folding simulations using the Rosetta modeling package (Rohl et al., 2004). By analyzing a large number of conformations sampled through a Monte Carlo procedure, we get a clearer picture of the preferred states of the protein and its dynamics. Our results show that the CTD may frequently adopt a structure with 3–5 helices and helix-turn-helix motifs in specific regions. Some of the best scoring structures show high similarity with the HMG-box-containing proteins previously used as templates by Bharath et al. Further clustering analysis of our results hints of a preferred set of conformations for the CTD of the linker histone. Comparison of these models with distances measured by FRET may help account for the distinct structures of the CTD observed upon binding to different macromolecular partners.     

Cover Picture: J. Biophotonics 9/2011

An example of Pallasea cancelloides – benthic Baikal amphipod employed for the studies of stress conditions. (Picture: D.V. Axenov‐Gribanov et al., pp. 619–626 in this issue)     

More insights into the CCN3/Connexin43 interaction complex and its role for signaling

Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc.     

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).     

In this issue: Proteomics 6/2009

In this issue of Proteomics you will find the following highlighted articles: Keeping up with the lung cancers You're in good company if you smoke and develop lung cancer. The World Health Organization estimates 1.2 million new cases occur every year. On the other hand, 1.1 million people die from it every year‐bummer. One reason for the high death rate is the frequent development of resistance to several of the most commonly used drugs simultaneously. Multiple drug resistance (MDR) is the major cause of chemotherapeutic failure. Keenan et al. explored the proteomic changes associated with MDR failure (adriamycin) in a cultured lung cancer cell line (DLKP) and several subtypes. Adriamycin normally kills by blocking replication at DNA gyrase and by generating reactive oxygen species that lead to apoptosis. Proteomes were examined by 2‐D DIGE. Approximately 80 proteins displayed quantitative shifts, 32 showed a correlation with resistance, 24 being linked positively to resistance, 6 correlated negatively. Some known targets did not appear on the 2‐D maps consistently. Keenan, J. et al., Proteomics 2009, 9, 1556‐1566. An image of spit Spitting images have been around for a long time. The phrase is possibly human‐kind's first recognition of genetically transmitted traits. Proteomic analysis of saliva has only developed recently. The question raised by Walz et al. here is “What is the possible contribution of saliva to the high level of infection by Helicobacter pylori?” H. pylori is known to have extracellular adhesins that bind to a number of salivary proteins. A convenient way to detect targets of adhesins was found to be incubating 1‐D and 2‐D PAGE Western blots with an overlay of whole H. pylori. Targets detected included mucins, sialic acid‐containing glycoproteins, fucose‐containing blood group antigens and each pair of salivary glands had a different binding pattern. Walz, A. et al., Proteomics 2009, 9, 1582‐1592. Mix'em up, folks Conventional analytical chemical identifications frequently yield a characteristic spectrum of peaks for particular compounds on particular instruments. Just look up the observed spectrum in the “library” of standard spectra for identification. It is not so simple for proteins. Because of the size of a potential proteomic peptide library and the diversity of instruments used, most often the observed spectrum is compared to a theoretical spectrum for a peptide of interest. Ahrné et al. combine the two for improved performance. First they run the spectrum of interest through an exhaustive proteome search program (Phenyx), then through a sensitive library search (SpectraST) of the highest scoring sequences in the previous Phenyx search plus a number of controls. In the first (relatively simple) test, Phenyx matched 362 spectra, SpectraST made 639 matches at the same error detection level. In a more complex test, Phenyx generated >1000 hits, SpectraST 1304 hits. Ahrné, E. et al., Proteomics 2009, 9, 1731‐1736.     

Cover Picture: Proteomics 6/2009

In this issue of Proteomics you will find the following highlighted articles: Keeping up with the lung cancers You're in good company if you smoke and develop lung cancer. The World Health Organization estimates 1.2 million new cases occur every year. On the other hand, 1.1 million people die from it every year‐bummer. One reason for the high death rate is the frequent development of resistance to several of the most commonly used drugs simultaneously. Multiple drug resistance (MDR) is the major cause of chemotherapeutic failure. Keenan et al. explored the proteomic changes associated with MDR failure (adriamycin) in a cultured lung cancer cell line (DLKP) and several subtypes. Adriamycin normally kills by blocking replication at DNA gyrase and by generating reactive oxygen species that lead to apoptosis. Proteomes were examined by 2‐D DIGE. Approximately 80 proteins displayed quantitative shifts, 32 showed a correlation with resistance, 24 being linked positively to resistance, 6 correlated negatively. Some known targets did not appear on the 2‐D maps consistently. Keenan, J. et al., Proteomics 2009, 9, 1556‐1566. An image of spit Spitting images have been around for a long time. The phrase is possibly human‐kind's first recognition of genetically transmitted traits. Proteomic analysis of saliva has only developed recently. The question raised by Walz et al. here is “What is the possible contribution of saliva to the high level of infection by Helicobacter pylori?” H. pylori is known to have extracellular adhesins that bind to a number of salivary proteins. A convenient way to detect targets of adhesins was found to be incubating 1‐D and 2‐D PAGE Western blots with an overlay of whole H. pylori. Targets detected included mucins, sialic acid‐containing glycoproteins, fucose‐containing blood group antigens and each pair of salivary glands had a different binding pattern. Walz, A. et al., Proteomics 2009, 9, 1582‐1592. Mix'em up, folks Conventional analytical chemical identifications frequently yield a characteristic spectrum of peaks for particular compounds on particular instruments. Just look up the observed spectrum in the “library” of standard spectra for identification. It is not so simple for proteins. Because of the size of a potential proteomic peptide library and the diversity of instruments used, most often the observed spectrum is compared to a theoretical spectrum for a peptide of interest. Ahrné et al. combine the two for improved performance. First they run the spectrum of interest through an exhaustive proteome search program (Phenyx), then through a sensitive library search (SpectraST) of the highest scoring sequences in the previous Phenyx search plus a number of controls. In the first (relatively simple) test, Phenyx matched 362 spectra, SpectraST made 639 matches at the same error detection level. In a more complex test, Phenyx generated >1000 hits, SpectraST 1304 hits. Ahrné, E. et al., Proteomics 2009, 9, 1731‐1736.     

Cover Picture: Proteomics 7/2009

In this issue of Proteomics you will find the following highlighted articles: Computing clusters and complexes At first glance, the structure of a cell looks like a semi‐random collection of proteins, lipids and nucleic acids. With the development of high‐throughput tools and bioinformatic procedures, we can begin to see some order in the chaos, including relationships that regulate cell functions (the interactome). Carbonell et al. looked at hubs, hot spots, interfaces, modules, complexes, binding site disorder, affinity and alanine scanning in developing a model for the energetics and specificity of protein‐protein interactions. They observed self‐segregation of binding sites by affinity, i.e. specific‐specific and promiscuous‐ promiscuous interactions between hubs are much higher than random association. Examples of low and high affinity energetics are discussed for cytochrome b, cdc42 GTPase, ubiquitin, and calmodulin‐dependent kinase. Calculated values were selectively validated for a reality check. Carbonell, P. et al., Proteomics 2009, 9, 1744‐1753. Pursuing the Plasmodium plague: understanding malaria through homology Plasmodium falciparum is a difficult organism to work with because of its complex life cycle: ring, trophozoite and schizont phases. From recent genome sequencing work, proteins/open reading frames can be selected by homology to look at possible elements of the plasmodium interactome. Wuchty et al. took on the challenge. Information was derived from reliable interaction experiments with S. cerevisiae, D. melanogaster, C. elegans, and E. coli. Homologies were determined by BlastP (all‐vs.‐all). Shared GO annotations were found which added to further understanding of the sparsely annotated parasite. Other parameters examined included Cluster Participation Coefficient, Kernel Density Function, K‐Clique Clustering, and (drum roll please) the Rich‐Club Coefficient. Using the InParanoid yeast database, they found over 1800 interactions among almost 700 yeast proteins. Pooling the four organisms gave 5000 interactions among 1900 proteins. There should be some interesting targets in there . . . Wuchty, S.et al., Proteomics 2009, 9, 1841‐1849 Race to the finish‐aging nerve vs. aging muscle Our image of a “senior citizen” often has a wobbling gait and sagging face. These are both in part the result of muscle atrophy. A good surgeon and $150 000 will get you the Joan Rivers look that should hold you into your 90's. But what about your legs? Tough luck for now. Capitanio et al., however, are looking at the relationship between muscle and nerve breakdown with age using proteomic tools. Studying the gastrocnemius muscle and the sciatic nerve of young (8 month) and older (22 month) rats, the authors found a number of coordinate morphological and metabolic changes in the deterioration of nerves and their linked muscles. Light and electron microscopy, 2‐D DIGE, ESI‐MS/MS MALDI‐TOF, Western immunoblots and immunocytochemistry were all brought to bear on the question. The results were a much clearer understanding of the mechanics of muscle aging. Capitanio, D. et al., Proteomics 2009, 9, 2004‐2020.