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2.
Cell Chem Biol ; 27(1): 32-40.e3, 2020 01 16.
Article in English | MEDLINE | ID: mdl-31653597

ABSTRACT

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.


Subject(s)
Drug Discovery , Drug Evaluation, Preclinical , Proprotein Convertase 9/metabolism , Proteolysis/drug effects , Serine Proteinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Humans , Ligands , Models, Molecular , Molecular Structure , Serine Proteinase Inhibitors/chemistry , Small Molecule Libraries/chemistry
3.
Cell Chem Biol ; 26(4): 584-592.e6, 2019 04 18.
Article in English | MEDLINE | ID: mdl-30745239

ABSTRACT

Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.


Subject(s)
Fluorescent Dyes/analysis , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Proteins/analysis , Rhodamines/analysis , Animals , Drosophila , Green Fluorescent Proteins/analysis , HeLa Cells , Humans , Recombinant Fusion Proteins/analysis , Staining and Labeling/methods
4.
Methods Mol Biol ; 1663: 65-78, 2017.
Article in English | MEDLINE | ID: mdl-28924659

ABSTRACT

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.


Subject(s)
Ceramides/metabolism , Fluorescent Dyes/metabolism , Golgi Apparatus/metabolism , Rhodamines/metabolism , Ceramides/chemical synthesis , Drug Stability , HeLa Cells , Humans , Imaging, Three-Dimensional , Microscopy, Fluorescence/methods
5.
Chem Commun (Camb) ; 53(80): 11036-11039, 2017 Oct 05.
Article in English | MEDLINE | ID: mdl-28937163

ABSTRACT

The effect of charged versus neutral N- and C-termini on the stability of the collagen triple helix was examined. Thermal denaturation studies at different pH with collagen model peptides showed that an ammonium group at the N-terminus destabilizes the triple helix more than a carboxylate at the C-terminus. A neutral carboxylic acid stabilizes the triple helix more than an amido moiety at the C-terminus.


Subject(s)
Collagen/chemistry , Carboxylic Acids/chemistry , Hydrogen-Ion Concentration , Protein Denaturation , Protein Stability , Protein Structure, Secondary , Temperature
6.
Nat Biotechnol ; 35(8): 773-780, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28671662

ABSTRACT

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.


Subject(s)
Cellular Structures/ultrastructure , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Nanotechnology/methods , HeLa Cells , Humans
7.
Mol Biol Cell ; 28(12): 1676-1687, 2017 Jun 15.
Article in English | MEDLINE | ID: mdl-28428254

ABSTRACT

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.


Subject(s)
ADP-Ribosylation Factor 1/physiology , Golgi Apparatus/physiology , ADP-Ribosylation Factor 1/genetics , ADP-Ribosylation Factor 1/metabolism , COP-Coated Vesicles/metabolism , Clathrin/metabolism , Coat Protein Complex I/metabolism , GTP Phosphohydrolases/metabolism , Golgi Apparatus/metabolism , Guanosine Triphosphate/metabolism , HeLa Cells , Humans , Hydrolysis , Intracellular Membranes/metabolism
8.
Chemistry ; 23(33): 7938-7944, 2017 Jun 12.
Article in English | MEDLINE | ID: mdl-28398637

ABSTRACT

(4S)- and (4R)-configured aminoproline (Amp) residues were used as pH-responsive probes to tune the thermal stability of collagen triple helices in acidic and basic environments. The different steric and stereoelectronic properties of amino versus ammonium groups lead to a switch of the ring pucker of Amp upon changing the pH. The choice of the position of Amp within collagen model peptides (CMPs) as well as the absolute configuration at C(4) of the pH-responsive probe allows for tuning of the stability of Amp-containing collagen triple helices over a broad range. Comparative quantum chemical calculations on the steric and stereoelectronic effects of amino and ammonium groups versus fluorine, hydroxy, chlorine, and methyl substituents support the experimental findings. The research also shows that substitution of the naturally occurring hydroxy group in collagen by electron-withdrawing groups with a larger hydration shell than that of the hydroxy group is not tolerated.

9.
Nat Commun ; 7: 10778, 2016 Mar 04.
Article in English | MEDLINE | ID: mdl-26940217

ABSTRACT

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.


Subject(s)
Heterocyclic Compounds, 4 or More Rings/chemistry , Luminescent Proteins , Microscopy, Fluorescence/methods , Nanotechnology/methods , Rhodamines/chemistry , Animals , COS Cells , Chlorocebus aethiops , HeLa Cells , Humans
10.
Chem Sci ; 6(12): 6725-6730, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-30154996

ABSTRACT

NMR spectroscopic studies with a series of proline derivatives revealed that the polarity of the environment has a significant effect on the trans : cis isomer ratio of Xaa-Pro bonds. Computational studies showed that this effect is due to differences in the overall dipole moments of trans and cis conformers. Comparisons between the conformational properties of amide and ester derivatives revealed an intricate balance between polarity effects and n → π* interactions of adjacent carbonyl groups. The findings have important implications for protein folding and signaling as well as the performance of proline-based stereoselective catalysts.

11.
Angew Chem Int Ed Engl ; 53(38): 10242-6, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25081303

ABSTRACT

We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid "tetrazine-click" reaction into Cer-SiR, a highly photostable "vital dye" that enables prolonged live-cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is nontoxic at concentrations as high as 2 µM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.


Subject(s)
Ceramides/chemistry , Coloring Agents/analysis , Coloring Agents/chemistry , Golgi Apparatus/metabolism , Cell Survival , Golgi Apparatus/enzymology , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Fluorescence
12.
Angew Chem Int Ed Engl ; 53(39): 10340-4, 2014 Sep 22.
Article in English | MEDLINE | ID: mdl-25088036

ABSTRACT

(4S)-Aminoproline is introduced as a pH-sensitive probe for tuning the conformational properties of peptides and proteins. The pH-triggered flip of the ring puckering and the formation/release of a transannular H bond were used to switch the formation of collagen triple helices on and off reversibly.


Subject(s)
Collagen/chemistry , Proline/analogs & derivatives , Circular Dichroism , Hydrogen Bonding , Hydrogen-Ion Concentration , Protein Structure, Secondary , Protein Structure, Tertiary , Stereoisomerism
13.
Bioorg Med Chem ; 21(12): 3565-8, 2013 Jun 15.
Article in English | MEDLINE | ID: mdl-23548629

ABSTRACT

Functionalized collagen is attractive for the development of synthetic biomaterials. Herein we present the functionalization of azidoproline containing collagen model peptides with various alkynes using click chemistry. The influence on the stability of the collagen triple helix of the stereochemistry of the introduced triazolyl prolines (4R or 4S), the position of their incorporation (Xaa or Yaa) and the substituents attached to them are shown. The results provide a useful guide for the optimal functionalization of collagen using click chemistry.


Subject(s)
Collagen/chemistry , Triazoles/chemistry , Alkynes/chemistry , Click Chemistry , Collagen/chemical synthesis , Protein Conformation , Protein Stability , Protein Structure, Secondary
14.
Chimia (Aarau) ; 67(12-13): 891-5, 2013.
Article in English | MEDLINE | ID: mdl-24594333

ABSTRACT

The article summarizes our research devoted to the development of functionalizable collagen using azidoproline-containing model peptides. 'Click chemistry' or Staudinger reduction followed by acylation provided facile access to a range of differently functionalized collagen model peptides bearing e.g. carbohydrates. The research provided not only insight into the factors that are responsible for the high conformational stability of collagen but also a guide in which positions functional moieties are tolerated without or by deliberately disturbing the supramolecular assembly of collagen. The presented research will be useful for the development of collagen-based materials.


Subject(s)
Azides/chemistry , Collagen/chemistry , Proline/chemistry , Protein Conformation
15.
J Am Chem Soc ; 134(41): 17117-24, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-22992124

ABSTRACT

The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.


Subject(s)
Collagen/chemistry , Collagen/chemical synthesis , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Stability , Thermodynamics
16.
Chemistry ; 18(40): 12783-93, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22930587

ABSTRACT

Certain Arctic and Antarctic ectotherm species have developed strategies for survival under low temperature conditions that, among others, consist of antifreeze glycopeptides (AFGP). AFGP form a class of biological antifreeze agents that exhibit the ability to inhibit ice growth in vitro and in vivo and, hence, enable life at temperatures below the freezing point. AFGP usually consist of a varying number of (Ala-Ala-Thr)(n) units (n=4-55) with the disaccharide ß-D-galactosyl-(1→3)-α-N-acetyl-D-galactosamine glycosidically attached to every threonine side chain hydroxyl group. AFGP have been shown to adopt polyproline II helical conformation. Although this pattern is highly conserved among different species, microheterogeneity concerning the amino acid composition usually occurs; for example, alanine is occasionally replaced by proline in smaller AFGP. The influence of minor and major sequence mutations on conformation and antifreeze activity of AFGP analogues was investigated by replacement of alanine by proline and glycosylated threonine by glycosylated hydroxyproline. The target compounds were prepared by using microwave-enhanced solid phase peptide synthesis. Furthermore, artificial analogues were obtained by copper-catalyzed azide-alkyne cycloaddition (CuAAC): propargyl glycosides were treated with polyproline helix II-forming peptides comprising (Pro-Azp-Pro)(n) units (n=2-4) that contained 4-azidoproline (Azp). The conformations of all analogues were examined by circular dichroism (CD). In addition, microphysical analysis was performed to provide information on their inhibitory effect on ice recrystallization.


Subject(s)
Antifreeze Proteins/chemistry , Antifreeze Proteins/chemical synthesis , Azides/chemistry , Carbohydrates/chemistry , Oligopeptides/chemistry , Oligopeptides/chemical synthesis , Proline/analogs & derivatives , Proline/chemistry , Circular Dichroism , Protein Conformation
17.
Org Biomol Chem ; 10(10): 1982-6, 2012 Mar 14.
Article in English | MEDLINE | ID: mdl-22266764

ABSTRACT

Click chemistry was used to introduce moieties as sterically demanding as monosaccharides into the Yaa position of collagen model peptides. The effect of different triazolyl derivatives as well as the configuration of the functionalized proline residue on the thermal stability of the collagen triple helices was examined.


Subject(s)
Click Chemistry , Collagen/chemistry , Peptides/chemistry , Triazoles/chemistry , Click Chemistry/methods , Protein Stability , Protein Structure, Secondary , Temperature
19.
J Am Chem Soc ; 132(40): 13957-9, 2010 Oct 13.
Article in English | MEDLINE | ID: mdl-20849115

ABSTRACT

The functionalizability and conformational properties of azidoproline (Azp)-containing collagen model peptides (CMPs) were studied. The results show that (4R)Azp has a similar stabilizing effect on the collagen triple helix as (4R)hydroxyproline and that functionalized CMPs are readily accessible by "click" chemistry. The resulting triazole-functionalized CMPs form stable triple helices, demonstrating that sterically demanding moieties in three symmetry-related positions in all strands are tolerated. The straightforward synthesis and facile functionalizability of the Azp-containing CMPs are intriguing for the development of functional collagen-based materials.


Subject(s)
Collagen/chemistry , Models, Molecular , Peptides/chemistry
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