Protein-Directed Self-Assembly of Gold Nanoparticles

AbstractWe have employed protein-protein interactions for controlled assembly of gold nanoparticles. Stoichiometric 1:1 protein:nanoparticle conjugates were prepared for proteins known to strongly interact with each other and these interactions were used to self-assemble nanoparticles. Mixing equivalent amounts of the antigen-nanoparticle and antibody-nanoparticle conjugates resulted in the formation of nanoparticle dimers with a yield of about 60%. Trimers (yield ∼30%) can be obtained by mixing 2:1 antigen-nanoparticle with 1:1 antibody-nanoparticle conjugates in a molar ratio of 1:2. The structures are destroyed at low pH when the antibody-antigen complex dissociates.

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Distance Controlled and Electrically Driven Photoluminescence Quench From Quantum Dot-Au Complexes

MRS Proceedings ◽

10.1557/proc-1257-o06-10 ◽

2010 ◽

Vol 1257 ◽

Author(s):

Zhitao Kang ◽

Jie Xu ◽

Dinal Andreasen ◽

Brent Karl Wagner

Keyword(s):

Gold Nanoparticles ◽

Protein Interactions ◽

Near Infrared ◽

Incident Light ◽

Gold Surface ◽

Electric Signal ◽

Protein Protein Interactions ◽

Quenching Effect ◽

Quenching Efficiency ◽

Pl Quenching

AbstractQuantum Dots (QDs) bound to gold nanoparticles have shown photoluminescence (PL) quenching dependent on distance between the two particles. The incident light from the QD couples to plasmon excitation of the metal when the frequencies of the light and the surface plasmon resonance (SPR) coincide, leading to a reduction in emitted PL in the system. The quenching effect of gold nanoparticles on QDs was used to study protein-protein interactions with the potential for drug screening applications. CdTe and CdHgTe QDs with emission wavelengths from 500˜900nm were synthesized and gold nanospheres and nanorods with controlled absorption in the visible and near-infrared (NIR) wavelength regions were prepared. The PL quenching of QD-Protein-Protein-Au complexes was studied as a function of Au concentration, QD size and protein type. A quenching efficiency of up to 90% was observed. The QD-Au complexes were also studied for electric potential sensing. The surface of the QDs was negatively charged due to thiol ligands capping. By applying a positive potential on the gold or gold nanoparticle attached substrate, the local electric field between the substrate and the statically charged QDs would pull the QDs closer to the gold surface and quench the QD PL. PL quenching of QD with Au was studied as a function of electric signal and QD type. In this methodology, electric signals were effectively converted to optical signals.

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DNA-mediated engineering of multicomponent enzyme crystals

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503533112 ◽

2015 ◽

Vol 112 (15) ◽

pp. 4564-4569 ◽

Cited By ~ 77

Author(s):

Jeffrey D. Brodin ◽

Evelyn Auyeung ◽

Chad A. Mirkin

Keyword(s):

Gold Nanoparticles ◽

Protein Interactions ◽

Building Blocks ◽

Inorganic Nanoparticles ◽

Protein Protein Interactions ◽

Dna Interactions ◽

Interparticle Interactions ◽

Crystalline Materials ◽

Catalytically Active ◽

Surface Chemistries

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.

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Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells

Journal of Virology ◽

10.1128/jvi.01910-07 ◽

2007 ◽

Vol 82 (1) ◽

pp. 60-70 ◽

Cited By ~ 218

Author(s):

Brent J. Ryckman ◽

Barb L. Rainish ◽

Marie C. Chase ◽

Jamie A. Borton ◽

Jay A. Nelson ◽

...

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Protein Interactions ◽

Low Ph ◽

Protein Protein Interactions ◽

Biologically Relevant ◽

Entry Pathway ◽

Virion Envelope ◽

Adenovirus Vectors

ABSTRACT The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.

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Selectivity of Protein Interactions along the Aggregation Pathway of α-Synuclein

10.1101/2021.01.29.428744 ◽

2021 ◽

Author(s):

André D. G. Leitão ◽

Paulina Rudolffi Soto ◽

Alexandre Chappard ◽

Akshay Bhumkar ◽

Dominic J. B. Hunter ◽

...

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Self Assembly ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Coincidence Detection ◽

Protein Protein Interactions ◽

Assembly Pathway ◽

Protein Partners ◽

Molecular Complexity

AbstractThe aggregation of α-SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease in neurons. Whilst α-synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of α-SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of α-SYN.In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of α-SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant α-SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with α-SYN monomers at detectable levels, and only a subset recognizes the oligomeric α-SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation.Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, α-SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different α-SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of α-SYN.

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Bioluminescence Resonance Energy Transfer System for Measuring Dynamic Protein-Protein Interactions in Bacteria

mBio ◽

10.1128/mbio.01050-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 6

Author(s):

Boyu Cui ◽

Yao Wang ◽

Yunhong Song ◽

Tietao Wang ◽

Changfu Li ◽

...

Keyword(s):

Energy Transfer ◽

Real Time ◽

Protein Interactions ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Low Ph ◽

Bacterial Cells ◽

Bioluminescence Resonance Energy Transfer ◽

Protein Protein Interactions ◽

Excitation Light

ABSTRACT Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. IMPORTANCE Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells, thus allowing the detection of protein-protein interactions in live bacterial cells. This BRET system added another useful tool to address important questions in microbiological studies.

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Designer protein assemblies with tunable phase diagrams in living cells

10.1101/2020.06.03.131433 ◽

2020 ◽

Author(s):

Meta Heidenreich ◽

Joseph M. Georgeson ◽

Emanuele Locatelli ◽

Lorenzo Rovigatti ◽

Saroj Kumar Nandi ◽

...

Keyword(s):

Phase Diagrams ◽

Protein Interactions ◽

Self Assembly ◽

Point Mutations ◽

Living Cells ◽

Protein Protein Interactions ◽

Material State ◽

Protein Components ◽

Novel Strategy

AbstractThe self-organization of proteins into specific assemblies is a hallmark of biological systems. Principles governing protein-protein interactions have long been known. However, principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remains challenging to characterize and understand. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies in living cells. We devise a novel strategy to map high-resolution phase diagrams in vivo, which provide mesoscale self-assembly signatures of our system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how point mutations that change their interaction affinity impact the phase diagram and material state of the assemblies in vivo. Both, the phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Applying our system to interrogate biological mechanisms of self-assembly, we find that co-translational protein binding suffices to recruit an mRNA to the designed micron-scale structures.

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Multiple Dimerizing Motifs Modulate the Dimerization of the Syndecan Transmembrane Domains

10.1101/2019.12.31.891929 ◽

2020 ◽

Author(s):

J. Chen ◽

F. Wang ◽

C. He ◽

S-Z. Luo

Keyword(s):

Protein Interactions ◽

Self Assembly ◽

Transmembrane Domains ◽

Integral Membrane Proteins ◽

Sequence Motifs ◽

Protein Protein Interactions ◽

Dynamics Simulations ◽

Considerable Complexity ◽

Shed Light ◽

Common Sequence

AbstractSyndecans(SDCs) are a family of four members of integral membrane proteins, which play important roles in cell-cell interactions. Dimerization/oligomerization generated by transmembrane domains (TMDs) appear to crucially regulate several functional behaviors of all syndecan members. The distinct hierarchy of protein-protein interactions mediated by the syndecan TMDs may give rise to considerable complexity in the functions of syndecans. The molecular mechanism of the different dimerization tendencies in each type of SDCs remains unclear. Here, the self-assembly process of syndecan TMD homodimers and heterodimers was studied in molecular details by molecular dynamics simulations. Our computational results showed that the SDC2 forms the most stable homodimer while the SDC1 TMD dimerizes weakly, which is consistent with previous experimental results. Detailed analysis suggests that instead of the conserved dimerizing motif G8XXXG12 in all four SDCs involved in homo- and hetero-dimerization of SDCs, the G3XXXA7 motif in SDC1 competes with the interface of G8XXXG12 and thus disturbs the SDC1 involved dimerization. The SDC3 which contains a G9XXXA13 motif, however, forms a more stable dimer than SDC1, indicating the complexity of the competing effect of the GXXXA motif. As GXXXG and GXXXA are two common sequence motifs in the dimerization of helices, our results shed light on the competing effect of multiple dimerizing motifs on the dimerization of transmembrane domains.

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Alcohol-Perturbed Self-Assembly of Tobacco Mosaic Virus Coat Protein

10.3762/bxiv.2022.2.v1 ◽

2022 ◽

Author(s):

Ismael Abu-Baker ◽

Amy Szuchmacher Blum

Keyword(s):

Coat Protein ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Protein Interactions ◽

Self Assembly ◽

Alcohol Concentration ◽

Protein Protein Interactions ◽

Virus Coat Protein ◽

Alcohol Water ◽

Virus Coat

Self-assembly of Tobacco mosaic virus coat protein is significantly altered in alcohol-water mixtures. Alcohol cosolvents stabilize the disk aggregate and prevent formation of helical rods at low pH. High alcohol content favours stacked disk assemblies and large rafts, while low alcohol concentration favours individual disks and short stacks. These effects appear to be caused by the hydrophobicity of the alcohol additive, with isopropyl alcohol having the strongest effect, and methanol the weakest. We hypothesize that alcohols interact with the hydrophobic faces of TMV-cp disks, thereby disrupting the protein-protein interactions between disks that are necessary to form helical rods.

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