Edward A. Lemke

Molecular Anatomy of a Trafficking Organelle

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.

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

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.

Interplay of alpha-synuclein binding and conformational switching probed by single-molecule fluorescence

We studied the coupled binding and folding of α-synuclein, an intrinsically disordered protein linked with Parkinsons 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 α-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple α-helical structures. α-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 α-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 α-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 α-synuclein.

A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures

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 ≈43 Å. This increased with denaturant in a noncooperative fashion to ≈63 Å, 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.

Amino Acids for Diels–Alder Reactions in Living Cells

The endeavour to perform tailored chemical reactions in the challenging environment of the intact cell delves deeply into the biological sciences. Requirements include strict bioorthogonality of the reactants and reactions that occur spontaneously and quickly in an aqueous environment or at the interface of membranes. Commonly used reactions that meet these criteria are Staudinger ligations and various forms of click chemistry. The most prominent among the latter is the Huisgen-type [3+2] cycloaddition between azides and alkynes. 2] Through the seminal work of the Bertozzi group, this reaction was stripped of its need for Cu catalysis by straining the alkyne group, thereby making this chemistry (termed strain-promoted alkyne–azide chemistry, SPAAC) viable in intact cells as well as in living animals. These reactions have been widely used to label molecules on cell surfaces and, in a few cases, inside the cell, for instance to label lipids, nucleotides, or carbohydrates. Another exciting click variant is strain-promoted inverse-electrondemand Diels–Alder cycloaddition (SPIEDAC), which can exhibit accelerated reaction rates by using strained reactants and furthermore is irreversible because of the loss of N2 (Scheme 1). This chemistry has been used in cells to label small molecules and is magnitudes faster than the classical Huisgen-type cycloadditions. To date, most biological applications of SPAAC or SPIEDAC do not involve modifications of proteins but instead alter cellular molecules that are not genetically encoded, such as metabolically incorporated sugars. Current tools for site-specific labeling of proteins within the cell use fluorescent protein fusions, self-alkylating protein additions, or high-affinity binding domains. The smallest size of artificial protein modifications currently available to introduce fluorescent labels are tetracysteine motifs consisting of six amino acids. Ideally the modification unit would be only a single artificial amino acid suitable for specific chemistry in cells. The introduction of such unnatural amino acids (UAAs) is possible by codon reassignment or by suppression of the Amber stop codon. For fluorescent labeling, genetically encoded azides can be used, but azides typically suffer from intracellular reduction. Furthermore, encoding the azide jeopardizes the design of a fluorogenic labeling scheme. 16] Fluorogenicity is of particular relevance for high-contrast imaging and super-resolution techniques, since dyes are turned on only after successful labeling, while nonspecifically attached dyes remain quenched. Rather than encoding azides, a more suitable approach is the use of an amino acid that carries the strained reactant, that is, a cyclooctyne group, thereby leaving the nitrogen-bearing reactants to serve as part of a fluorogenic probe. If suppression of the Amber stop codon is used, a single residue in a specified protein can then be replaced with the strained alkyne. This type of protein labeling by using an artificially introduced cyclooctyne amino acid and fluorogenic azides Scheme 1. a) Structures of strained alkene and alkyne UAAs. b) Reaction scheme showing orthogonality and cross-reactivity of SPIEDAC and SPAAC with fluorogenic tetrazine-functionalized dyes (gray sphere) and azide-functionalized dyes (green sphere). Dyes coupled to tetrazine are only fluorescent (green) after successful labeling.

Single-molecule biophysics: at the interface of biology, physics and chemistry

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.

Genetically Encoded Copper-Free Click Chemistry

The ability to visualize biomolecules within living specimen by engineered fluorescence tags has become a major tool in modern biotechnology and cell biology. Encoding fusion proteins with comparatively large fluorescent proteins (FPs) as originally developed by the Chalfie and Tsien groups is currently the most widely applied technique.[1] As synthetic dyes typically offer better photophysical properties than FPs, alternative strategies have been developed based on genetically encoding unique tags such as Halo and SNAP tags, which offer high specificity but are still fairly large.[2] Small tags like multi-histidine[3] or multi-cysteine motifs[4] may be used to recognize smaller fluorophores, but within the cellular environment they frequently suffer from poor specificity as their basic recognition element is built from native amino acid side chains. Such drawbacks may be overcome by utilizing bioorthogonal chemistry that relies on coupling exogenous moieties of non-biological origin under mild physiological conditions. A powerful chemistry that fulfils these requirements is the Huisgen type (3+2) cycloaddition between azides and alkynes (a form of click chemistry[5]). By utilizing supplementation-based incorporation techniques and click reactions Beatty et al. coupled azide derivatized dyes to Escherichia coli expressing proteins bearing linear alkynes.[6] However, this azide–alkyne cycloaddition required copper(I) as a catalyst (CuAAC), which strongly reduces biocompatibility (but see Ref. [7]). This limitation has been overcome by Bertozzi and co-workers, who showed that the “click” reaction readily proceeds when utilizing ring-strained alkynes as a substrate[8] and since then this strain-promoted azide–alkyne cycloaddition (SPAAC) has found increasing applications in labeling, for example, carbohydrates,[9] nucleotides,[10] and lipids.[11] Further expanding the potential of this approach, Ting and co-workers engineered a lipolic acid ligase which ligates a small genetically encoded recognition peptide to a cylcooctyne-containing substrate. In a second step the incorporated cyclooctyne moiety then functioned as a specific site for labeling in cells.[12]

Cell type‐specific nuclear pores: a case in point for context‐dependent stoichiometry of molecular machines

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.

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

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.

In situ structural analysis of the human nuclear pore complex

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.