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Lemke GroupPublications

Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy.
Nikic, I., Plass, T., Schraidt, O., Szymanski, J., Briggs, J.A., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2014 Jan 28. doi: 10.1002/anie.201309847.
The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.
Europe PMC

Continuous throughput and long-term observation of single-molecule FRET without immobilization.
Tyagi, S., Vandelinder, V., Banterle, N., Fuertes, G., Milles, S., Agez, M. & Lemke, E.A.
Nat Methods. 2014 Jan 19. doi: 10.1038/nmeth.2809.
We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.
Europe PMC

Fourier ring correlation as a resolution criterion for super-resolution microscopy.
Banterle, N., Bui, K.H., Lemke, E.A. & Beck, M.
J Struct Biol. 2013 Sep;183(3):363-7. doi: 10.1016/j.jsb.2013.05.004. Epub 2013May 16.
Optical nanoscopy techniques using localization based image reconstruction, also termed super-resolution microscopy (SRM), have become a standard tool to bypass the diffraction limit in fluorescence light microscopy. The localization precision measured for the detected fluorophores is commonly used to describe the maximal attainable resolution. However, this measure takes not all experimental factors, which impact onto the finally achieved resolution, into account. Several other methods to measure the resolution of super-resolved images were previously suggested, typically relying on intrinsic standards, such as molecular rulers, or on a priori knowledge about the specimen, e.g. its spatial frequency content. Here we show that Fourier ring correlation provides an easy-to-use, laboratory consistent standard for measuring the resolution of SRM images. We provide a freely available software tool that combines resolution measurement with image reconstruction.
Europe PMC

A new family of bioorthogonally applicable fluorogenic labels.
Herner, A., Nikic, I., Kallay, M., Lemke, E.A. & Kele, P.
Org Biomol Chem. 2013 May 28;11(20):3297-306. doi: 10.1039/c3ob40296g.
Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical properties were elaborated by experimental and theoretical investigations. Of the newly synthesized fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic performance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while their "clicked" triazole congeners showed intense fluorescence. One of these labels showed a very large Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluorogenic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen of possible fluorogenic tags will enable the generation of libraries of such tags in the future.
Europe PMC

Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines.
Ori, A., Banterle, N., Iskar, M., Andres-Pons, A., Escher, C., Khanh Bui, H., Sparks, L., Solis-Mezarino, V., Rinner, O., Bork, P., Lemke, E.A. & Beck, M.
Mol Syst Biol. 2013 Mar 19;9:648. doi: 10.1038/msb.2013.4.
To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.
Europe PMC

Facilitated aggregation of FG nucleoporins under molecular crowding conditions.
Milles, S., Huy Bui, K., Koehler, C., Eltsov, M., Beck, M. & Lemke, E.A.
EMBO Rep. 2013 Feb;14(2):178-83. doi: 10.1038/embor.2012.204. Epub 2012 Dec 14.
Intrinsically disordered and phenylalanine-glycine-rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high-resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.
Europe PMC

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.
Lukinavicius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Correa IR, J.r, Luo, Z.G., Schultz, C., Lemke, E.A., Heppenstall, P., Eggeling, C., Manley, S. & Johnsson, K.
Nat Chem. 2013 Feb;5(2):132-9. doi: 10.1038/nchem.1546. Epub 2013 Jan 6.
The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.
Europe PMC

What precision-protein-tuning and nano-resolved single molecule sciences can do for each other.
Milles, S. & Lemke, E.A.
Bioessays. 2013 Jan;35(1):65-74. doi: 10.1002/bies.201200094. Epub 2012 Nov 13.
While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints.
Europe PMC

Genetically Encoded Click Chemistry for Single-Molecule FRET of Proteins.
Tyagi, S. & Lemke, E.A.
Methods Cell Biol. 2013;113:169-87. doi: 10.1016/B978-0-12-407239-8.00009-4.
Single molecule Fluorescence Resonance Energy Transfer (FRET) has been widely applied to study structure, function and dynamics of complex biological systems. Labeling of proteins at specific positions with fluorescent dyes is a challenging and key step for any single molecule FRET measurement. Genetic code expansion has facilitated site specific incorporation of unnatural amino acids into proteins. These unnatural amino acid bears bioorthognal functional groups that provide opportunity to install a unique chemical handle into proteins. Propargyllysine is an unnatural amino acid which, when incorporated into a protein, can be exploited to attach commercially available fluorescent azide dyes through copper-catalyzed alkyne-azide cycloaddition click reaction (also known as click reaction). We describe here an optimized strategy to combine synthesis of propargyllysine, its genetic incorporation in the protein and click reaction to site-specifically label the protein with azide derivative of Alexa(R) 488. Later the protein is labeled at unique cysteine residue via maleimide coupling chemistry with acceptor Alexa(R) 594 dye to yield double labeled protein as required for any single molecule FRET experiments.
Europe PMC

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.
Milles, S., Koehler, C., Gambin, Y., Deniz, A.A. & Lemke, E.A.
Mol Biosyst. 2012 Oct;8(10):2531-4. doi: 10.1039/c2mb25135c.
Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.
Europe PMC

Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation.
Borrmann, A., Milles, S., Plass, T., Dommerholt, J., Verkade, J.M., Wiessler, M., Schultz, C., van Hest, J.C., van Delft, F.L. & Lemke, E.A.
Chembiochem. 2012 Sep 24;13(14):2094-9. doi: 10.1002/cbic.201200407. Epub 2012Sep 3.
Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.
Europe PMC

Conserved features of intermediates in amyloid assembly determine their benign or toxic states.
Krishnan, R., Goodman, J.L., Mukhopadhyay, S., Pacheco, C.D., Lemke, E.A., Deniz, A.A. & Lindquist, S.
Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11172-7. doi:10.1073/pnas.1209527109. Epub 2012 Jun 27.
Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Abeta, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Abeta, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.
Europe PMC

Amino acids for diels-alder reactions in living cells.
Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2012 Apr 23;51(17):4166-70. doi: 10.1002/anie.201108231.Epub 2012 Mar 30.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site-specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans-cyclooctene units that react with tetrazine derivatives in an inverse-electron-demand Diels-Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide-cyclooctyne click reaction (right).
Europe PMC

Click strategies for single-molecule protein fluorescence.
Milles, S., Tyagi, S., Banterle, N., Koehler, C., Vandelinder, V., Plass, T., Neal, A.P. & Lemke, E.A.
J Am Chem Soc. 2012 Mar 21;134(11):5187-95. Epub 2012 Mar 5.
Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molec studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.
Europe PMC

Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153.
Milles, S. & Lemke, E.A.
Biophys J. 2011 Oct 5;101(7):1710-9. doi: 10.1016/j.bpj.2011.08.025.
Nucleoporins (Nups), which are intrinsically disordered, form a selectivity filter inside the nuclear pore complex, taking a central role in the vital nucleocytoplasmic transport mechanism. These Nups display a complex and nonrandom amino-acid architecture of phenylalanine glycine (FG)-repeat clusters and intra-FG linkers. How such heterogeneous sequence composition relates to function and could give rise to a transport mechanism is still unclear. Here we describe a combined chemical biology and single-molecule fluorescence approach to study the large human Nup153 FG-domain. In order to obtain insights into the properties of this domain beyond the average behavior, we probed the end-to-end distance (R(E)) of several approximately 50-residues long FG-repeat clusters in the context of the whole protein domain. Despite the sequence heterogeneity of these FG-clusters, we detected a reoccurring and consistent compaction from a relaxed coil behavior under denaturing conditions (R(E)/R(E,RC) = 0.99 +/- 0.15 with R(E,RC) corresponding to ideal relaxed coil behavior) to a collapsed state under native conditions (R(E)/R(E,RC) = 0.79 +/- 0.09). We then analyzed the properties of this protein on the supramolecular level, and determined that this human FG-domain was in fact able to form a hydrogel with physiological permeability barrier properties.
Europe PMC

Principles for designing fluorescent sensors and reporters.
Lemke, E.A. & Schultz, C.
Nat Chem Biol. 2011 Jul 18;7(8):480-3. doi: 10.1038/nchembio.620. Europe PMC

Genetically encoded copper-free click chemistry.
Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2011 Apr 18;50(17):3878-81. doi:10.1002/anie.201008178. Epub 2011 Mar 23. Europe PMC

Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing.
Gambin, Y., VanDelinder, V., Ferreon, A.C., Lemke, E.A., Groisman, A. & Deniz, A.A.
Nat Methods. 2011 Mar;8(3):239-41. doi: 10.1038/nmeth.1568. Epub 2011 Feb 6.
We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein alpha-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.
Europe PMC

2.05 - Förster Resonance Energy Transfer.
Lemke, E.A. & Deniz, A.A.
In Comprehensive Nanoscience and Technology, edited by David L. Andrews, Gregory D. Scholes and Gary P. Wiederrecht, Academic Press, Amsterdam, 2011, Pages 127-151
There is wide interest in Förster (fluorescence) resonance energy transfer (FRET) as a noninvasive and high-sensitivity nanoscale ruler with a broad array of implications and applications ranging from basic quantum physics to nanotechnology and biology. This chapter provides a selected overview of the field. Basic theoretical and experimental concepts in FRET are first presented, followed by a few selected advanced applications in biology. This is followed by a discussion of the development of single-molecule FRET and its applications that have unfolded over the last decades. Extensions, variants, and future directions of FRET for ensemble and single-molecule studies are also discussed.

Site-specific labeling of proteins for single-molecule FRET measurements using genetically encoded ketone functionalities.
Lemke, E.A.
Methods Mol Biol. 2011;751:3-15. doi: 10.1007/978-1-61779-151-2_1.
Studies of protein structure and function using single-molecule fluorescence resonance energy transfer (smFRET) benefit dramatically from the ability to site-specifically label proteins with small fluorescent dyes. Genetically encoding the unnatural amino acid (UAA) p-acetylphenylalanine is an efficient way to introduce commercially available fluorescent tags with high yield and specificity. This protocol describes the expression in Escherichia coli of proteins containing this UAA in response to the amber stop codon TAG. Proteins were purified with high yield and subsequently labeled with the hydroxylamine derivative of Alexa Fluor(R) 488 functioning as a fluorescent donor dye. The proteins were then labeled via maleimide coupling chemistry at a unique cysteine with the acceptor dye Alexa Fluor(R) 594 to yield a dual-labeled protein ready for subsequent smFRET observation.
Europe PMC

Precision control of cellular pathways with light.
Lemke, E.A.
Chembiochem. 2010 Sep 3;11(13):1825-7. Europe PMC

Microfluidic device for single-molecule experiments with enhanced photostability.
Lemke, E.A., Gambin, Y., Vandelinder, V., Brustad, E.M., Liu, H.W., Schultz, P.G., Groisman, A. & Deniz, A.A.
J Am Chem Soc. 2009 Sep 30;131(38):13610-2.
A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents.
Europe PMC

Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae.
Lee, H.S., Guo, J., Lemke, E.A., Dimla, R.D. & Schultz, P.G.
J Am Chem Soc. 2009 Sep 16;131(36):12921-3.
Here, we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be genetically incorporated into proteins in yeast with excellent selectivity and efficiency by means of an orthogonal tRNA/aminoacyl-tRNA synthetase pair. This small, environmentally sensitive fluorophore was site-specifically incorporated into Escherichia coli glutamine binding protein and used to directly probe local structural changes caused by ligand binding. The small size of Anap and the ability to introduce it by simple mutagenesis at defined sites in the proteome make it a useful local probe of protein structure, molecular interactions, protein folding, and localization.
Europe PMC

Direct single-molecule observation of a protein living in two opposed native structures.
Gambin, Y., Schug, A., Lemke, E.A., Lavinder, J.J., Ferreon, A.C., Magliery, T.J., Onuchic, J.N. & Deniz, A.A.
Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10153-8. doi:10.1073/pnas.0904461106. Epub 2009 Jun 8.
Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (smFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function.
Europe PMC

Interplay of alpha-synuclein binding and conformational switching probed by single-molecule fluorescence.
Ferreon, A.C., Gambin, Y., Lemke, E.A. & Deniz, A.A.
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5645-50. Epub 2009 Mar 17.
We studied the coupled binding and folding of alpha-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of alpha-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple alpha-helical structures. Alpha-synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive alpha-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the alpha-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of alpha-synuclein.
Europe PMC

A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.
Brustad, E.M., Lemke, E.A., Schultz, P.G. & Deniz, A.A.
J Am Chem Soc. 2008 Dec 31;130(52):17664-5.
A general strategy for the site-specific dual-labeling of proteins for single-molecule fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-containing fluorophore with high yield (>95%) and specificity. This methodology was used to construct dual-labeled T4 lysozyme variants, allowing the study of T4 lysozyme folding at single-molecule resolution. The presented strategy is anticipated to expand the scope of single-molecule protein structure and function studies.
Europe PMC

Single-molecule biophysics: at the interface of biology, physics and chemistry.
Deniz, A.A., Mukhopadhyay, S. & Lemke, E.A.
J R Soc Interface. 2008 Jan 6;5(18):15-45.
Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo.
Europe PMC

Control of protein phosphorylation with a genetically encoded photocaged amino acid.
Lemke, E.A., Summerer, D., Geierstanger, B.H., Brittain, S.M. & Schultz, P.G.
Nat Chem Biol. 2007 Dec;3(12):769-72. Epub 2007 Oct 28.
We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.
Europe PMC

Immune complexes of auto-antibodies against A beta 1-42 peptides patrol cerebrospinal fluid of non-Alzheimer's patients.
Henkel, A.W., Dittrich, P.S., Groemer, T.W., Lemke, E.A., Klingauf, J., Klafki, H.W., Lewczuk, P., Esselmann, H., Schwille, P., Kornhuber, J. & Wiltfang, J.
Mol Psychiatry. 2007 Jun;12(6):601-10. Epub 2007 Feb 6.
The diagnostic potential of large A beta-peptide binding particles (LAPs) in the cerebrospinal fluid (CSF) of Alzheimer's dementia (AD) patients and non-AD controls (nAD) was evaluated. LAPs were detected by confocal spectroscopy in both groups with high inter-individual variation in number. Molecular imaging by confocal microscopy revealed that LAPs are heterogeneous superaggregates that could be subdivided morphologically into four main types (LAP 1-4). LAP-4 type, resembling a 'large chain of pearls', was detected in 42.1% of all nAD controls but it was virtually absent in AD patients. LAP-4 type could be selectively removed by protein A beads, a clear indication that it contained immunoglobulins in addition to beta-amyloid peptides (A beta 1-42). We observed a close correlation between LAPs and immunoglobulin G (IgG) concentration in CSF in controls but not in AD patients. Double labeling of LAPs with anti-A beta and anti-IgG antibodies confirmed that LAP-4 type consisted of A beta and IgG aggregates. Our results assign a central role to the immune system in regulating A beta1-42 homeostasis by clustering this peptide in immunocomplexes.
Europe PMC

A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures.
Mukhopadhyay, S., Krishnan, R., Lemke, E.A., Lindquist, S. & Deniz, A.A.
Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2649-54. Epub 2007 Feb 13.
The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was approximately equal to 43 A. This increased with denaturant in a noncooperative fashion to approximately equal to 63 A, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.
Europe PMC

Molecular anatomy of a trafficking organelle.
Takamori, S., Holt, M., Stenius, K., Lemke, E.A., Gronborg, M., Riedel, D., Urlaub, H., Schenck, S., Brugger, B., Ringler, P., Muller, S.A., Rammner, B., Grater, F., Hub, J.S., De Groot, B.L., Mieskes, G., Moriyama, Y., Klingauf, J., Grubmuller, H., Heuser, J., Wieland, F. & Jahn, R.
Cell. 2006 Nov 17;127(4):831-46.
Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.
Europe PMC

Single synaptic vesicle tracking in individual hippocampal boutons at rest and during synaptic activity.
Lemke, E.A. & Klingauf, J.
J Neurosci. 2005 Nov 23;25(47):11034-44.
How synaptic vesicles move within central nervous synapses to their docking sites at the plasma membrane is widely discussed in synaptic physiology. This question is especially difficult to investigate in the small hippocampal boutons, which themselves can slowly move during observation in primary cell culture. Here, we describe a single particle tracking method using dual fluorescent dye labels that enabled us to visualize the movements of a single vesicle and the respective synaptic bouton simultaneously during resting conditions and stimulation. We found vesicle mobility to be very low in the absence of stimulation, in line with previous studies. Interestingly, mobility was also found to be low during synaptic activity. We found that vesicles labeled preferentially via early, late, and spontaneous endocytotic mechanisms behaved similarly at rest and during stimulation.
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Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy.
Jordan, R., Lemke, E.A. & Klingauf, J.
Biophys J. 2005 Sep;89(3):2091-102. Epub 2005 Jun 24.
Not much is known about the mobility of synaptic vesicles inside small synapses of the central nervous system, reflecting a lack of methods for visualizing these dynamics. We adapted confocal spot detection with fluctuation analysis to monitor the mobility of fluorescently labeled synaptic vesicles inside individual boutons of cultured hippocampal neurons. Using Monte Carlo simulations we were able to propose a simple quantitative model that can describe vesicle mobility in small hippocampal boutons under resting conditions and different pharmacological treatments. We find that vesicle mobility in a time window of 20 s can be well described by caged diffusion (D approximately 5 x 10(-5) microm(2)/s, cage sizes of approximately 50 nm). Mobility can be upregulated by phosphatase blockage and increased further by actin disruption in a dose-dependent manner. Inhibition of the myosin light chain kinase slows down vesicle mobility 10-fold, whereas other kinases like protein kinase C (PKC), A (PKA), and calmodulin kinase II (caMKII) do not affect mobility in unstimulated boutons.
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Identification and mutational studies of conserved amino acids in the outer membrane receptor protein, FepA, which affect transport but not binding of ferric-enterobactin in Escherichia coli.
Chakraborty, R., Lemke, E.A., Cao, Z., Klebba, P.E. & van der Helm, D.
Biometals. 2003 Dec;16(4):507-18.
Many gram-negative bacteria produce and excrete siderophores, which complex iron with high affinity in the environment. The ferric siderophore complexes are transported across the outer membrane by receptor proteins. This process requires energy and is TonB dependent and must involve conformational changes in the receptor proteins to allow the transport of the ferric siderophores from the extracellular binding site to the periplasm. There is a large variety in the structures, molecular weights and charges among the siderophores. It was therefore realized that when the sequences of the many different receptor proteins were compared, simultaneously, all identities and close similarities, found in this manner, could only be due to residues involved in the conformational changes and transport mechanism, common to all the proteins, and not be due to the specificity of ligand recognition. Once the crystal structures of FepA, FhuA and FecA became available, it was immediately clear that the sequence similarities which were found in the simultaneous alignment, were all localized in a few structural domains, which are identical in the three structures and can therefore be expected to be maintained in all the proteins in this family. One of these domains, tentatively named the lock region, consists of 10 residues with a central quadrupole formed by two arginines and two glutamates, from the plug region and the beta barrel. We mutated several of these residues in FepA. All showed normal binding in quantitative binding studies. Some showed normal transport as well, however, the majority showed moderate to severe defective transport with ferric enterobactin. The results therefore show the validity of the hypothesis that the simultaneous sequence alignment will select the residues involved in the transport function of the receptor proteins. In addition the results allow to relate the severity of the transport deficiency to be correlated with the structure of the lock region while it is also possible to propose a function of this region in the conformational changes of the protein during the transport of the ligand from the binding site to the periplasm.
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