Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.     

Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity

Single-molecule FRET has been widely used for monitoring protein-nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0-3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG-I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.     

Cross-correlation analysis of X-ray photon correlation spectroscopy to extract rotational diffusion coefficients

Coefficients for translational and rotational diffusion characterize the Brownian motion of particles. Emerging X-ray photon correlation spectroscopy (XPCS) experiments probe a broad range of length scales and time scales and are well-suited for investigation of Brownian motion. While methods for estimating the translational diffusion coefficients from XPCS are well-developed, there are no algorithms for measuring the rotational diffusion coefficients based on XPCS, even though the required raw data are accessible from such experiments. In this paper, we propose angular-temporal cross-correlation analysis of XPCS data and show that this information can be used to design a numerical algorithm (Multi-Tiered Estimation for Correlation Spectroscopy [MTECS]) for predicting the rotational diffusion coefficient utilizing the cross-correlation: This approach is applicable to other wavelengths beyond this regime. We verify the accuracy of this algorithmic approach across a range of simulated data.

The analysis of Brownian motion of different types of particles is a classic problem in research fields such as molecular biology and materials science. For a dilute suspension, collisions from the solvent particles lead to random reposition and reorientation that can be decomposed into translational and rotational diffusion. These two types of diffusion can be characterized by coefficients, namely, the translational diffusion coefficient Dt and rotational diffusion coefficient Dr in two dimensions and the corresponding 3×3 tensor in three dimensions. Knowledge of these parameters provides insight into the structure and dynamic properties of the particles, opening the door for understanding functions and transport process of proteins (1), synthesis and stability of materials (2), biomolecular reactions (3), etc.As an emerging X-ray scattering technique, X-ray photon correlation spectroscopy (XPCS) is able to probe length scale down to nanometers and time scale from below microseconds to hours and is well-suited for studying dynamics of disordered systems, of which analyzing diffusion is a critical informative characteristic. In XPCS experiments, samples are illuminated by partially coherent X-ray beams, and time series of scattering images are collected by detectors. (See Fig. 1 for an illustration of the XPCS experiments.) The inhomogeneity of the electron density leads to spatial variation of the brightness of the images, which is called a ”speckle pattern,” and the fluctuation of the electron density leads to the temporal variation of the speckle patterns that contains valuable information of the sample dynamics, which can be revealed by analyzing these collected, time-dependent images.Open in a separate windowFig. 1.Schematic illustration of the XPCS experiments. The translation and rotation of the particles within the scattering volume leads to variation of the speckle patterns shown on the right. (While the grainy, noise-like texture makes these images appear visually similar, the proposed algorithm is able to detect and analyze the contained variations.)One of the most well-known tools for analyzing the images is the temporal autocorrelation function g2, which depends on the second-order degree of coherence (4); see, for example, refs. 5–7. While translational diffusion coefficients Dt can be determined through g2, to the best of our knowledge, there is no current algorithm to extract Dr from XPCS data.The objective of this paper is to present a methodology for calculating the rotational diffusion coefficient Dr in the two-dimensional (2D) case using XPCS data. By exploiting the more detailed angular-temporal cross-correlation function, we are able to discover more information than the autocorrelation function g2. Information about this cross-correlation function can be related to the static electron density of individual particles and the rotational diffusion coefficient Dr. To estimate Dr from the proposed cross-correlation, we first introduce a mathematical model and related set of equations, whose solution corresponds to the rotational diffusion. We then design a numerical algorithm, “Multi-Tiered Estimation for Correlation Spectroscopy (MTECS),” based on an approach introduced here, which solves the relevant equations by following a multitiered iterative projection (M-TIP) philosophy, first introduced in ref. 8. In particular, we construct several operators and apply them in an iteration to efficiently map the cross-correlation data into a form that can then be processed by the algorithm to converge to the solution of the underlying equations.While we mainly discuss XPCS in this paper, the developed methodology does not rely upon the specific range of the wavelength essentially, so that it can be generalized to other experiment techniques, such as dynamic light scattering. Though dynamic light scattering has been applied to measure the rotational diffusion (9, 10), these methods are limited by their requirements of the type and structure of the particles. The MTECS algorithm requires almost no prior assumption about particle structure, but can take advantage of such information if a priori known. MTECS is robust against noisy data, as it includes additional filtering methods applied to the input cross-correlation data.     

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract–binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5′ and 3′ ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5′ and the 3′ pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.     

Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy

Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment.It is axiomatic that the quality of water is essential for human health (1). The increasing worldwide contamination of freshwater systems with thousands of industrial and natural chemical compounds is one of the key environmental problems facing humanity today, where pathogens in water cause more than 2 million deaths annually (2). With more than one-third of the accessible and renewable freshwater used for industrial, agricultural, and domestic applications, pollution from these activities leaves water sources contaminated with numerous synthetic and geogenic compounds (2, 3). In addition, natural disasters can result in large-scale disruptions of infrastructure, resulting in compromised water quality. Diarrheal disease caused from such disasters may be a major contributor to overall morbidity and mortality rates (4). Thus, the cleanliness and safety of public water sources has prompted researchers to look for rapid and sensitive indicators of water quality. Whereas most water filtering systems are quite efficient in removing large-size contaminants, smaller particles frequently pass through. These contaminants are often poorly soluble in water, thus, present in quantities of less than 1 nM. Here, we demonstrate femtomolar detection of urobilin, a biomarker found in human and animal waste in water.Modern analytical tools have become extremely efficient in the detection and analysis of chemical compounds. For example, liquid chromatography coupled with detection by tandem mass spectrometry has been commonly used for detection of trace pharmaceuticals and other wastewater-derived micropollutants (5). Although such methods are very powerful in identifying trace pollutants, cost prohibits their widespread use by environmental researchers and, most importantly, prevents real-time analysis of water quality (6). Other techniques using bench top gas chromatography–mass spectrometry have also been demonstrated as viable methods for detection of basic pharmaceuticals with reduced cost (7). Despite this, these methods are still cost prohibitive, can hardly be used in field studies, and are unlikely to ever be used for real-time quality control.In addition to pharmaceutical and other synthetic pollutants such as pesticides, animal and human waste (i.e., feces, urine) is an enormous source of water contamination that can be found in both recreational and source waters. These discarded products, when released into water, can carry a variety of diseases such as polio, typhoid, and cholera (8). In extreme cases pollution of an ecosystem can result in environmental crises, such as devastation to the aquatic population, red-tide blooms, as well as beach closings. Molecular methods based on polymerase chain reactions are commonly used to monitor viral, bacterial, and protozoan pathogens in wastewater (9). Microbiological indicators such as fecal coliforms, Escherichia coli and Etherococci, are the indicators most commonly used to analyze and evaluate the level of fecal contamination. However, the suitability of these indicators has been questioned (10), and it takes a substantial time from the extraction of water sample for analysis to the moment when the results are ready.An alternative indicator that has been shown to be helpful in detection of waste in water supplies is urobilin (11). Urobilin is one of the final byproducts of hemoglobin metabolism, and is excreted in both the urine and feces of many mammals, including humans and common livestock (cows, horses, and pigs) (12). In addition, as urobilin can be indicative of disease such as hepatic dysfunction, or jaundice, an ultrasensitive technique for detection and quantification of this biomarker in solution has both diagnostic and environmental applications.Urobilin detection in solution has previously been demonstrated using the formation of a phosphor group from the combination of urobilins and zinc ions (13). Normal heme catabolism results in the production of bilirubin, a red product, which is then broken down into two end products, stercobilin, the bile pigment found in fecal material, and urobilin, the yellow pigment found in urine. Both urobilin and stercobilin have been shown to be viable biomarkers for detection of fecal pollution levels in rivers (14).Fluorescent detection of urobilin in urine has been demonstrated based on Schlesinger’s reaction in which an urobilinogen–zinc chelation complex exhibits a characteristic green fluorescence when excited by blue light (15). Methods for detection of urobilinoids using high-performance liquid chromatography with a reversed-phase column and an ultraviolet detector have also been presented (16); however, the initial sensitivity of this method proved insufficient for clinical analysis. Miyabara and coworkers reported an increase in detection sensitivity of this methodology, but only to detection levels of 1.5 nM (13), where efficient excitation and collection of the fluorescent signal remained the limiting factor. Traditional epiillumination fluorescence spectroscopy systems use an objective lens to focus excitation light into the sample and collect the fluorescence emission. In such a configuration, the signal generated is limited to the focal volume of the optics. In addition, the generated signal is diffusive in nature; only a small fraction of the total emitted light is collected. Because only a small volume of a sample can be probed at any given time with such a configuration, detection of subnanomolar concentrations remains difficult as these measurements are akin to single molecule detection. Thus, a method that could allow for probing a larger volume of a sample while also providing means for collecting more of the fluorescence emission could greatly enhance the ability to detect subnanomolar concentrations of urobilin.     

Cis- and trans-membrane interactions of synaptotagmin-1

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca(2+) triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca(2+)-dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca(2+)-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca(2+)-dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis- and trans-binding, which may play an important modulatory role in synaptic transmission.     

Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.     

Single-molecule force spectroscopy distinguishes target binding modes of calmodulin

The eukaryotic signaling protein calmodulin (CaM) can bind to more than 300 known target proteins to regulate numerous functions in our body in a calcium-dependent manner. How CaM distinguishes between these various targets is still largely unknown. Here, we investigate fluctuations of the complex formation of CaM and its target peptide sequences using single-molecule force spectroscopy by AFM. By applying mechanical force, we can steer a single CaM molecule through its folding energy landscape from the fully unfolded state to the native target-bound state revealing equilibrium fluctuations between numerous intermediate states. We find that the prototypical CaM target sequence skMLCK, a fragment from skeletal muscle myosin light chain kinase, binds to CaM in a highly cooperative way, while only a lower degree of interdomain binding cooperativity emerges for CaMKK, a target peptide from CaM-dependent kinase kinase. We identify minimal binding motifs for both of these peptides, confirming that affinities of target peptides are not exclusively determined by their pattern of hydrophobic anchor residues. Our results reveal an association mode for CaMKK in which the peptide binds strongly to only partially Ca²⁺-saturated CaM. This binding mode might allow for a fine-tuning of the intracellular response to changes in Ca²⁺ concentration.     

Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry

Among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems. During the signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between Ffh (the protein component of SRP) and FtsY (the SRP receptor) is known to be essential for the efficiency and fidelity of this process. In this work, we studied the Escherichia coli and Thermus aquaticus Ffh.FtsY complexes by using chemical cross-linking and tandem mass spectrometry to identify nine intermolecular cross-linked peptides. This information was used in conjunction with a previously undescribed model-building approach that combines geometric restraint optimization with macromolecular docking. The resulting model of the Ffh.FtsY complex is in good agreement with the crystal structure solved shortly thereafter. Intriguingly, four of the cross-linked pairs involve the M domain of Ffh, which is absent from the crystal structure, providing previously undocumented experimental evidence that the M domain is positioned in close proximity to the Ffh.FtsY interface in the complex.     

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.     

N-terminal domain of human Hsp90 triggers binding to the cochaperone p23

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial. Here, we characterized the Apo and ATP states of the 170-kDa human Hsp90 full-length protein by NMR spectroscopy in solution, and we elucidated the mechanism of the inhibition of its ATPase by its cochaperone p23. We assigned isoleucine side chains of Hsp90 via specific isotope labeling of their δ-methyl groups, which allowed the NMR analysis of the full-length protein. We found that ATP caused exclusively local changes in Hsp90's N-terminal nucleotide-binding domain. Native mass spectrometry showed that Hsp90 and p23 form a 22 complex via a positively cooperative mechanism. Despite this stoichiometry, NMR data indicated that the complex was not fully symmetric. The p23-dependent NMR shifts mapped to both the lid and the adenine end of Hsp90's ATP binding pocket, but also to large parts of the middle domain. Shifts distant from the p23 binding site reflect p23-induced conformational changes in Hsp90. Together, we conclude that it is Hsp90's nucleotide-binding domain that triggers the formation of the Hsp90(2)p23(2) complex. We anticipate that our NMR approach has significant impact on future studies of full-length Hsp90 with cofactors and substrates, but also for the development of Hsp90 inhibiting anticancer drugs.     

Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM

Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross–pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.     

Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO(2) nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO(2) to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO(2) into the catalyzed membrane provided an eightfold reduction in ROS generation rate.     

Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.     

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of ~44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K_i~10.1 μM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.     

Recognition of trimethyllysine by a chromodomain is not driven by the hydrophobic effect

Posttranslational modifications of histone proteins regulate gene expression via complex protein-protein and protein-DNA interactions with chromatin. One such modification, the methylation of lysine, has been shown to induce binding to chromodomains in an aromatic cage [Nielsen PR, et al. (2002) Nature 416:103-107]. The binding generally is attributed to the presence of cation-pi interactions between the methylated lysine and the aromatic pocket. However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a beta-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation-pi interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.