Nanoscale click-reactive scaffolds from peptide self-assembly.

Background Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, as the amyloid state is accessible to a large number of proteins via misfolding, assembly of peptides already conjugated to proteins is limited to certain cargo species. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. Results Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen azidoalkyne cycloaddition, one of the class of “click” reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. Discussion Although previous studies have produced peptides that can both form amyloid fibrils and undergo “click”-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation

Spontaneous CO release from Ru(II)(CO)2-protein complexes in aqueous solution, cells, and mice.

We demonstrate that Ru(II)(CO)2-protein complexes, formed by the reaction of the hydrolytic decomposition products of [fac-RuCl(κ(2)-H2NCH2CO2)(CO)3] (CORM-3) with histidine residues exposed on the surface of proteins, spontaneously release CO in aqueous solution, cells, and mice. CO release was detected by mass spectrometry (MS) and confocal microscopy using a CO-responsive turn-on fluorescent probe. These findings support our hypothesis that plasma proteins act as CO carriers after in vivo administration of CORM-3. CO released from a synthetic bovine serum albumin (BSA)-Ru(II)(CO)2 complex leads to downregulation of the cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in cancer cells. Finally, administration of BSA-Ru(II)(CO)2 in mice bearing a colon carcinoma tumor results in enhanced CO accumulation at the tumor. Our data suggest the use of Ru(II)(CO)2-protein complexes as viable alternatives for the safe and spatially controlled delivery of therapeutic CO in vivo.We thank the FCT, the EU, and the EPSRC for funding. G.J.L.B. is a Royal Society University Research Fellow.This is the final published version. It first appeared at http://onlinelibrary.wiley.com/doi/10.1002/anie.201409344/abstract

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site

Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Biotechnology and Biological Sciences Research CouncilFunder: Centre for Misfolding Diseases, University of CambridgeFunder: Wellcome TrustBiomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region

A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter

Raw data supporting 'Purification of recombinant α-Synuclein: a comparison of commonly used protocols'

Raw data from MTT assays, kinetic aggregation assays, densitometry, absorption and mass spec experiments

Mining 2:2 Complexes from 1:1 Stoichiometry: Formation of Cucurbit[8]uril−Diarylviologen Quaternary Complexes Favored by Electron-Donating Substituents

A 1:1 binding stoichiometry of a host−guest complex need not consist of a single host and guest. Diarylviologens containing electron-donating substituents complexed with cucurbit[8]uril (CB[8]) in a 1:1 stoichiometry exhibit abnormally large binding enthalpies compared to typical enthalpy changes observed for 1:1 binary complexes. Here, several CB[8]-mediated host−guest complexes, which were previously reported as 1:1 binary complexes, are verified to be 2:2 quaternary complexes by a combination of isothermal titration calorimetry, $^1$H, NOESY, and ROESY NMR, and ion mobility mass spectrometry, clearly indicating a binding motif of two partially overlapping diarylviologens held in place with two CB[8] molecules. Formation of 2:2 quaternary complexes is favored by electron-donating substituents, while electron-withdrawing substituents typically result in 1:1 binary complexes. The stacking of two highly conjugated diarylviologens in one quaternary motif affords the complexes enhanced conductance when considered as a single-molecular conductor. Moreover, an additional conducting signal previously observed for this “supramolecular” conductor can be readily understood with our 2:2 complexation model, corresponding to a parallel conductance pathway. Therefore, a 2:2 quaternary complex model grants a greater understanding of such supramolecular complexes, enabling the design of engineered, hierarchical structures and functional materials.The authors thank the Leverhulme Trust (project: ‘Natural material innovation for sustainable living’), the Marie Curie FP7 SASSYPOL ITN (607602) programme, and EPSRC (EP/ L504920/1) for funding

Multimeric complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5:insights into the structure and assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

[Image: see text] Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5(NTD)) but not to Cul2(NTD). The structure of the monomeric ASB9–EloBC–Cul5(NTD) quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems