Cross-linked Pairs Research Articles

Prediction of peptide mass spectral libraries with machine learning.

The recent development of machine learning methods to identify peptides in complex mass spectrometric data constitutes a major breakthrough in proteomics. Longstanding methods for peptide identification, such as search engines and experimental spectral libraries, are being superseded by deep learning models that allow the fragmentation spectra of peptides to be predicted from their amino acid sequence. These new approaches, including recurrent neural networks and convolutional neural networks, use predicted in silico spectral libraries rather than experimental libraries to achieve higher sensitivity and/or specificity in the analysis of proteomics data. Machine learning is galvanizing applications that involve large search spaces, such as immunopeptidomics and proteogenomics. Current challenges in the field include the prediction of spectra for peptides with post-translational modifications and for cross-linked pairs of peptides. Permeation of machine-learning-based spectral prediction into search engines and spectrum-centric data-independent acquisition workflows for diverse peptide classes and measurement conditions will continue to push sensitivity and dynamic range in proteomics applications in the coming years.

Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen.

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

Accurate Retention Time Prediction Based on Monolinked Peptide Information to Confidently Identify Cross-Linked Peptides.

Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome-wide level mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the retention time as an extra filter, the false positive rate can be reduced by around 86% with a sensitivity loss of 10%. The method combined with DTSS (T-DTSS) not only benefits improving identification confidence but also leads to lower cutoff scores and facilitates substantially increasing inter-cross-link identification. T-DTSS was successfully applied to the identification of inter-cross-links obtained from Escherichia coli cell lysate cross-linked by a newly synthesized enrichable cross-linker, pDSBE. The approach can be applicable to both cleavable and noncleavable methods.

Design of Metal-Organic Framework Templated Materials Using High-Throughput Computational Screening.

The ability to crosslink Metal-Organic Frameworks (MOFs) has recently been discovered as a flexible approach towards synthesizing MOF-templated “ideal network polymers”. Crosslinking MOFs with rigid cross-linkers would allow the synthesis of crystalline Covalent-Organic Frameworks (COFs) of so far unprecedented flexibility in network topologies, far exceeding the conventional direct COF synthesis approach. However, to date only flexible cross-linkers were used in the MOF crosslinking approach, since a rigid cross-linker would require an ideal fit between the MOF structure and the cross-linker, which is experimentally extremely challenging, making in silico design mandatory. Here, we present an effective geometric method to find an ideal MOF cross-linker pair by employing a high-throughput screening approach. The algorithm considers distances, angles, and arbitrary rotations to optimally match the cross-linker inside the MOF structures. In a second, independent step, using Molecular Dynamics (MD) simulations we quantitatively confirmed all matches provided by the screening. Our approach thus provides a robust and powerful method to identify ideal MOF/Cross-linker combinations, which helped to identify several MOF-to-COF candidate structures by starting from suitable libraries. The algorithms presented here can be extended to other advanced network structures, such as mechanically interlocked materials or molecular weaving and knots.

Assessment of a new dual effective combo polymer structure for separation of lysozyme from hen egg white

Water-soluble O-carboxymethyl chitosan (O-CMCS) Schiff base granules were prepared, and Cu2+ ions were decorated. Then, dual effective combo cryogel structures were prepared by free radical polymerization using Cu2+-O-CMCS granules as embedding agent. 2-Hydroxyethyl methacrylate and N,N′-methylene-bis-acrylamide were used as monomer and cross-linker pair, respectively. Prepared combo structures were characterized by FTIR, SEM and swelling tests measurements, and investigated for lysozyme adsorption. Maximum lysozyme adsorption was observed in phosphate buffer of pH 8.0 as 103.3 mg/g with initial lysozyme concentration of 3 mg/mL. On the other hand, the most efficient adsorption was carried out at 0.5 mL/min, at 25 °C. It was also observed that combo cryogels could be repeatedly used for adsorption and desorption cycles of lysozyme molecules. The purification efficiency of adsorbent was highlighted as 83.4% with 88.2% purification yield from hen egg white via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

Allosteric Regulation of Rod Photoreceptor Phosphodiesterase 6 (PDE6) Elucidated by Chemical Cross-Linking and Quantitative Mass Spectrometry

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ2) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ2, and Pαβγ2–cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.

Hybrid magnetite-gold nanoparticles as bifunctional magnetic-plasmonic systems: three representative cases.

Hybrid systems based on magnetite and gold nanoparticles have been extensively used as bifunctional materials for bio- and nano-technology. The properties of these composites are assumed to be closely related to the magnetite to gold mass ratio and to the geometry of the resulting hetero-structures. To illustrate this, we compare and analyze the optical and magnetic properties of core-shell, dumbbell-like dimers and chemical cross-linked pairs of magnetite and gold nanoparticles in detail. We explore how the combination of gold with magnetite can lead to an improvement of the optical properties of these systems, such as tunability, light scattering enhancement or an increase of the local electric field at the interface between magnetic and plasmonic constituents. We also show that although the presence of gold might affect the magnetic response of these hybrid systems, they still show good performance for magnetic applications; indeed the resulting magnetic properties are more dependent on the NP size dispersion. Finally, we identify technological constraints and discuss prospective routes for the development of further magnetic-plasmonic materials.

Influence of n-alkyl ester groups on efficiency of crosslinking for methacrylate monomers copolymerized with EGDMA: Experiments and Monte Carlo simulations of reaction kinetics and sol–gel structure

We present the results of applying a computationally efficient Monte Carlo (MC) method to simulate the reaction kinetics and molecular architecture development during free-radical, bulk copolymerizations of a homologous series of methacrylate monomers with ethylene glycol dimethacrylate. The overall objective of the work was to determine the extent to which the ester side chains of the methacrylate monomers hinder chain-end radical propagation reactions through the pendent vinyl groups of the crosslinking monomer. We have determined that this steric hindrance is quite significant and increases with ester side chain length. This is expressed by a reduced reactivity parameter, ΨC,X, that is different for each monomer–crosslinker pair. With this single kinetic parameter adjustment, the MC model was able to effectively predict the reaction kinetics, gel points, and sol−gel fractions throughout the entire reactions, including, to a limited extent, the solvent swelling ratio of the gel for two different levels of crosslinker.

Dramatic Domain Rearrangements of the Cyanobacterial Orange Carotenoid Protein upon Photoactivation.

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection.

Abstract 3236: Development of a novel cross-linking strategy to identify distinct proteasome subtypes

Abstract Introduction: This research was conducted to develop a practical method to identify distinct proteasome subtypes. Approval of the proteasome inhibitors bortezomib and carfilzomib for clinical use has validated the proteasome as an anticancer target. These inhibitors target two well-recognized proteasome subtypes, the constitutive proteasome and the immunoproteasome, which comprise distinct sets of catalytic subunits. Additional proteasome subtypes containing mixtures of constitutive and immunoproteasome catalytic subunits have more recently been identified. These intermediate proteasome subtypes display unique proteolytic activity profiles and play important roles in the production of certain tumor antigens. However, much regarding their regulation, functions, and relevance as drug targets remains unknown, largely due to limitations in current experimental methods used to differentiate between proteasome subtypes. We have therefore developed bifunctional activity-based probes capable of cross-linking different catalytic subunit pairs within individual proteasome complexes. These probes allow a straightforward readout of proteasome catalytic subunit composition and facilitate functional studies of distinct proteasome subtypes. Experimental Procedures: Bifunctional proteasome probes were synthesized by coupling pairs of proteasome inhibitors via linkers of varying lengths and compositions. Positions for inhibitor derivatization were selected based on computational modeling, and linkers were chosen based on distances between the active sites of each targeted subunit pair. The ability of these compounds to cross-link distinct catalytic subunit pairs in both purified proteasomes and in cancer cell lysates was visualized by western blotting analysis. Competition assays with subunit-selective proteasome inhibitors were employed to confirm the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional proteasome probes were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results demonstrated the ability of these compounds to cross-link multiple pairs of proteasome catalytic subunits, allowing the composition of distinct proteasome subtypes present within cancer cells to be evaluated. Conclusions: We have successfully developed a set of bifunctional proteasome probes for analysis of proteasome catalytic subunit composition. These probes will be used to elucidate the unique functions of distinct proteasome subtypes present within cancer cells. Citation Format: Kimberly C. Carmony, Do-Min Lee, Lalit Kumar Sharma, Jieun Park, Kyung-Bo Kim, Wooin Lee. Development of a novel cross-linking strategy to identify distinct proteasome subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2014-3236

Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.

Abstract Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells. Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes. Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells. Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231

Solving the Recalcitrant Crystal Structure of Group II Chaperonin TRiC/CCT by Mass Spectrometry and Sentinel Correlation Analysis

Eukaryotic group II Chaperonin TRiC or CCT is a 0.95 megadalton protein complex that is essential for the correct and efficient folding of cytosolic polypeptides. The closed form is a 16 nm sphere made of two hemi-spherical rings of 8 subunits (∼550 residues/subunit) that rotate to open a central folding chamber. In eukaryotes, 8 different genes encode the subunits of this ATP-powered nanomachine. The high sequence identity of subunits made the 40,320 (=8 factorial) possible arrangements indistinguishable in previous cryo-electron microscopy and crystallographic analysis.First we use cross-linking, mass spectrometry and combinatorial homology modeling. We react bovine TRiC under native conditions with a lysine-specific cross-linker, follow up with trypsin digestion, and use mass spectrometry to identify 63 cross-linked pairs providing distance restraints. Independently of the cross-link set, we construct all 40,320 homology models of the TRiC particle. When we compared each model with the cross-link set, we discovered that one model is significantly more compatible than any other model. Bootstrapping analysis confirms that this model is 10 times more likely to result from this cross-link set than the next best-fitting model.Second, we re-examine the 3.8 A resolution X-ray data of yeast TRiC. Our method of Sentinel Correlation Analysis (SCA) exhaustively tests all 2,580,460 possible models. This unbiased analysis singles out with overwhelming significance one model, which is fully consistent with our previous biochemical data and refines to a much lower Rfree value than reported previously with the same X-ray data. With four-fold averaging, our structure reveals remarkably resolved details of the unique conformation of each subunit, and suggests a mechanism for the initiation of transition to the open state. More generally, we expect SCA to resolve ambiguity in future low-resolution crystallographic studies.

Protein flexibility is key to cisplatin crosslinking in calmodulin.

Chemical crosslinking in combination with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) has significant potential for studying protein structures and protein-protein interactions. Previously, cisplatin has been shown to be a crosslinker and crosslinks multiple methionine (Met) residues in apo-calmodulin (apo-CaM). However, the inter-residue distances obtained from nuclear magnetic resonance structures are inconsistent with the measured distance constraints by crosslinking. Met residues lie too far apart to be crosslinked by cisplatin. Here, by combining FTICR MS with a novel computational flexibility analysis, the flexible nature of the CaM structure is found to be key to cisplatin crosslinking in CaM. It is found that the side chains of Met residues can be brought together by flexible motions in both apo-CaM and calcium-bound CaM (Ca₄-CaM). The possibility of cisplatin crosslinking Ca₄-CaM is then confirmed by MS data. Therefore, flexibility analysis as a fast and low-cost computational method can be a useful tool for predicting crosslinking pairs in protein crosslinking analysis and facilitating MS data analysis. Finally, flexibility analysis also indicates that the crosslinking of platinum to pairs of Met residues will effectively close the nonpolar groove and thus will likely interfere with the binding of CaM to its protein targets, as was proved by comparing assays for cisplatin-modified/unmodified CaM binding to melittin. Collectively, these results suggest that cisplatin crosslinking of apo-CaM or Ca₄-CaM can inhibit the ability of CaM to recognize its target proteins, which may have important implications for understanding the mechanism of tumor resistance to platinum anticancer drugs.

Structural Kinetics of Troponin during Activation of Skeletal Muscle Fibers by Photolysis of Nitrophenyl-EGTA (NP-EGTA)

Mutants of troponin C (TnC) with bifunctional rhodamine (BR) cross-linking pairs of cysteine residues 56 and 63 or 96 and 103, along the C- and E-helices respectively, were exchanged into demembranated fibers from rabbit psoas muscle. Changes in the orientation of the two helices following a sudden increase in [Ca2+] elicited by photolysis of NP-EGTA were monitored by polarized fluorescence. The second- and fourth-rank order parameters, and respectively, that describe the orientation distribution of the BR dipoles with respect to the muscle fiber axis were calculated from the polarized fluorescence intensities. 5 ms after photolysis, when force was only 3% of maximum, the changes in and were 90% complete for the C-helix and 65% for the E-helix. The results reveal the kinetics of the first steps in the signaling pathway in the troponin complex: the [Ca2+] jump induces a fast structural change in the N-terminal lobe of TnC, containing the regulatory Ca2+ sites near the C helix, followed by a structural change in its C-lobe (containing the E helix) that is still faster than force generation. When active force development was inhibited by N-benzyl-p-toluene-sulphonamide (BTS), the kinetics and amplitude of the orientation change in the C-helix were not affected, whereas the orientation change in the E-helix was reduced. The effect of BTS on the kinetics also revealed a component of the E-helix orientation change that is synchronous with force development and accounts for the amplitude decrease, suggesting that myosin head binding to actin completes the structural change of the C-lobe initially triggered by calcium binding to the N-lobe.Supported by Medical Research Council and Wellcome Trust, UK.

Role of Structural Water in the Stabilization of the KcsA C-Type Inactivated Selectivity Filter: Evidence from High-Resolution Structures

A first glimpse into the structural changes underlying the interconversion between conductive and non-conductive C-type inactivated forms of the K+ channel selectivity filter (SF) at modest resolution was obtained from crystal structure of open-inactivated KcsA (O/I) [1]. On the basis of these structures, we developed a set of double cysteine crosslinking pairs that sharply increase the resolution of KcsA crystals with its gate trapped in the open configuration. Here, we present the crystal structures of KcsA locked-open (O) together with mutations that stabilize either the C-type inactivated filter, Y82A (O/I); or the fully conductive filter, E71A (O/O) @ 2.3 and 2.1 A, respectively. Comparison of these structures helps identify a novel water network (H2O_1, H2O_2 and H2O_3) behind the channel SF that stabilizes the O/I conformation of the channel. H2O_1 interacts with the backbone carbonyl groups of V76 and E71 from a neighboring subunit and with H2O_2+H2O_3 within the same subunit. In KcsA, the presence of the E71 residue on the channel pore helix is essential to drive the channel to the O/I state while providing a high-dielectric media suitable for the formation of this water-molecule network behind the SF. The intersubunit nature of this interaction, which is present only in the O/I state, provides a cogent structural explanation for the cooperative character of the C-type inactivation process in K+ channels. Additionally, we propose that the formation of a similar water-molecules mediated H-Bond network behind the SF of other K+ channels is essential to stabilize the O/I state.1. Cuello, L.G., et al. Nature, 2010. 466(7303): p. 203-8.

Role of Direct Interactions between the Histone H4 Tail and the H2A Core in Long Range Nucleosome Contacts

In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To determine whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide cross-linking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand association but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Additionally, we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermolecular self-association.

Changes in the Conformation of Troponin C on Activation of Skeletal Muscle

Skeletal muscle contraction is regulated by calcium-dependent changes in the structure and thin-filament location of troponin and tropomyosin. The structural changes in the isolated calcium-binding subunit of troponin (TnC) are well characterized, but those of TnC in the native thin filament are much less clear. We measured the in situ orientation of the C-terminal lobe of TnC (CTnC) by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteines at TnC residues 96-103, 116-123, 132-139, and 119-135. Each BR-labeled TnC was exchanged into single permeablized fibers from rabbit psoas muscle, and polarized fluorescence from the BR-TnCs was measured during relaxation and maximal calcium activation. The orientation distribution of CTnC with respect to the thin filament axis was calculated by maximum entropy analysis using the in vitro structure of CTnC in the troponin core complex (Vinogradova et al. (2005) PNAS102:5038-5043). The peak angle between E helix of CTnC and the filament axis was 49° in relaxed muscle and 64° during maximal activation. Comparison with the results of our previous study of the orientation of the N-terminal lobe of TnC (Ferguson et al. (2003) Mol. Cell11: 865-874) suggests that the central D/E helix of TnC is bent by about 30° in relaxed muscle and becomes straight during maximal activation.Supported by MRC (UK).

Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

On Parallel and Antiparallel Topology of a Homodimeric Multidrug Transporter

The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ion-coupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity (negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.

Nanoscale Features of Fibronectin Fibrillogenesis Depend on Protein-Substrate Interaction and Cytoskeleton Structure

Cell-reorganized fibronectin layers on polymer films providing a gradation of the binding strength between protein and substrate were analyzed by combined fluorescence and scanning force microscopy. The nanoscale fibronectin patterns exhibited paired parallel fibrils with characteristic spacings of 156, 233, 304, and 373 nm. These spacings depend on the interaction of fibronectin with the substrate: at enhanced fibronectin-substrate anchorage the cells form larger stress fibers, which are assembled by α-actinin cross-linked pairs of actin filaments subunits at the focal adhesions. A ubiquitous repeating unit of ∼71 nm was found within these characteristic distances. We conclude that the dimensions of the actin stress fibers reflect the binding strength of fibronectin to the polymer substrate and act—in turn—as a template for the reorganization of fibronectin into surface-bound nanofibrils with characteristic spacings. This explanation was confirmed by data showing the α-actinin/fibronectin colocalization.