Bifunctional Reagents for the Crosslinking of Proteins

Fasold, H.; Klappenberger, Jürgen; Meyer, C. T.; Remold, Heinz

doi:10.1002/anie.197107951

Cited by 45 publications

(36 citation statements)

References 16 publications

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“…It would be exceptionally difficult on the other hand to isolate the structural or functional effects of a particular crosslink among all other crosslinks formed by a chemical compound. Lastly, chemical crosslinkers may bind and thus disrupt the hydrophobic core of proteins because of their hydrophobicity (1).…”

Section: Fig 4 Mapping Of Crosslinks By Lc-ms/ms and Crossfinder Smentioning

confidence: 99%

“…Crosslinking methods have been powerful tools for decades to obtain information about the structural organization of proteins (1). Under the assumption that crosslinks only form between neighboring subunits, rough topological models of protein complexes could be delineated (2).…”

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confidence: 99%

“…A number of years ago however, Schultz and Chin introduced a technique allowing the unnatural amino acid p-benzoyl-p-phenylalanine (Bpa) 1 to be genetically encoded (17). Bpa is incorporated into the polypeptide chain by the ribosome during translation (Fig.…”

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confidence: 99%

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Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Forné¹,

Ludwigsen

Imhof

et al. 2012

Molecular & Cellular Proteomics

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We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes. Structural information about this family is scarce, hampering our mechanistic understanding of chromatin remodeling. Making use of cells that harbor a special tRNA/aminoacyl-tRNA synthetase pair, several residues within the ATPase domain of ISWI were individually substituted with the UV-reactive unnatural amino acid p-benzoyl-p-phenylalanine. Intramolecular crosslinks could be mapped with amino acid precision by high resolution tandem mass spectrometry and the novel bioinformatic tool "Crossfinder." Most crosslinks were fully consistent with published crystal structures of ISWIrelated ATPases. A subset of crosslinks, however, disagreed with the conformations previously captured in crystal structures. We built a structural model using the distance information obtained from the crosslinks and the structure of the closest crystallized relative, Chd1. The model shows the ATPase lobes strongly rotated against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state. The minimal requirements for solubility and protein amounts make our approach ideal for studying structures and conformations of proteins that are not amenable to conventional structural techniques. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.012088, 1-11, 2012.Crosslinking methods have been powerful tools for decades to obtain information about the structural organization of proteins (1). Under the assumption that crosslinks only form between neighboring subunits, rough topological models of protein complexes could be delineated (2). With advances in MS instrumentation and computational analysis of the MS data, it became possible to precisely determine the residues involved in the crosslink (3-5). Crosslinks provide constraints on the through-space distance of the attachment sites, and this information can aid structure prediction (6, 7), can distinguish protein conformations (8), and can identify the interaction surface between proteins (9, 10) and between proteins and their ligands (11). Crosslinking-MS-based methods are widely applicable as they only require microgram quantities of protein, are not limited by protein size or solubility and are relatively tolerant against sample heterogeneity. Moreover, crosslinking approaches can also be applied in vivo (12-16).Two strategies are employed to crosslink proteins. Most simply, a bifunctional chemical compound is added to the protein sample. Alternatively, an amino acid with a photoreactive side chain moiety is site-specifically incorporated into the polypeptide during synthesis. The latter method has several advantages (see also Discussion). M...

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Section: Fig 4 Mapping Of Crosslinks By Lc-ms/ms and Crossfinder Smentioning

confidence: 99%

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confidence: 99%

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Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Forné¹,

Ludwigsen

Imhof

et al. 2012

Molecular & Cellular Proteomics

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“…However, the decrease of σ c (Table 1) is a clear indicator for an increase of the particle diameter, and we regard it as rather unlikely to originate from aggregation. Aging of the samples for 4 days leads to a minor increase in relaxation time which might be caused most probably by further growth of the opsonisation layer or possibly by crosslinking between surface proteins [29,30].…”

Section: Resultsmentioning

confidence: 99%

Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

Weidner¹,

Gräfe²,

Lühe³

et al. 2016

Nano Online

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Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40°C. For incubation temperatures above 50°C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona composition.

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“…Nonisomerizable bi-functional crosslinkers have for decades been applied in molecular and structural biology as 'molecular rulers' during protein function and protein assembly (Fasold et al 1971;Ji 1983). In 2000, Woolley and colleagues introduced crosslinkers that contain a central AB moiety for the optical control of the secondary structure of peptides.…”

Section: Pxs: Molecular Tweezersmentioning

confidence: 99%

Flipping the Photoswitch: Ion Channels Under Light Control

McKenzie

Sánchez-Romero

Janovjak

2015

Advances in Experimental Medicine and Biology

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Nature has incorporated small photochromic molecules, colloquially termed 'photoswitches', in photoreceptor proteins to sense optical cues in phototaxis and vision. While Nature's ability to employ light-responsive functionalities has long been recognized, it was not until recently that scientists designed, synthesized and applied synthetic photochromes to manipulate biological processes with the temporal and spatial resolution of light. Ion channels in particular have come to the forefront of proteins that can be put under the designer control of synthetic photochromes.Photochromic ion channel controllers are comprised of three classes, photochromic soluble ligands (PCLs), photochromic tethered ligands (PTLs) and photochromic crosslinkers (PXs), and in each class ion channel functionality is controlled through reversible changes in photochrome structure. By acting as light-dependent ion channel agonists, antagonist or modulators, photochromic controllers effectively converted a wide range of ion channels, including voltage-gated ion channels, 'leak channels', tri-, tetra-and pentameric ligand-gated ion channels, and temperaturesensitive ion channels, into man-made photoreceptors. Control by photochromes is reversible, unlike in the case of 'caged' compounds, and non-invasive with high spatial precision, unlike pharmacology and electrical manipulation. Here, we introduce design principles of emerging photochromic molecules that act on ion channels and discuss the impact that these molecules are beginning to have on ion channel biophysics and neuronal physiology.

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Bifunctional Reagents for the Crosslinking of Proteins

Cited by 45 publications

References 16 publications

Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

Flipping the Photoswitch: Ion Channels Under Light Control

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