Engineering of a Small Protein Scaffold To Recognize Sulfotyrosine with High Specificity

2021 ◽  
Author(s):  
Justin Lawrie ◽  
Sean Waldrop ◽  
Anya Morozov ◽  
Wei Niu ◽  
Jiantao Guo
Keyword(s):  
High Specificity ◽  
Protein Scaffold ◽  
Small Protein

scholarly journals Near-Atomic Cryo-EM Imaging of a Small Protein Displayed on a Designed Scaffolding System

2017 ◽  
Author(s):  
Yuxi Liu ◽  
Shane Gonen ◽  
Tamir Gonen ◽  
Todd O. Yeates
Keyword(s):  
Nucleic Acid ◽  
Protein Design ◽  
Single Particle ◽  
Tight Binding ◽  
Protein Subunit ◽  
Large Protein ◽  
Protein Scaffold ◽  
Small Protein ◽  
Cubic Symmetry ◽  
Typical Size

AbstractCurrent single particle electron cryo-microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size amenable to cryo-EM. Using a rigid continuous alpha-helical linker, we connect a small 17 kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5 to 5 Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryoEM to proteins of typical size in the cell.Significance statementNew electron microscopy methods are making it possible to view the structures of large proteins and nucleic acid complexes at atomic detail, but the methods are difficult to apply to molecules smaller than about 50 kDa, which is larger than the size of the average protein in the cell. The present work demonstrates that a protein much smaller than that limit can be successfully visualized when it is attached to a large protein scaffold designed to hold 12 copies of the attached protein in symmetric and rigidly defined orientations. The small protein chosen for attachment and visualization can be modified to bind to other diverse proteins, opening up a new avenue for imaging cellular proteins by cryo-EM.

Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

2020 ◽  
Vol 15 (12) ◽  
pp. 2000078 ◽  
Author(s):  
Kyra See ◽  
Tetsuya Kadonosono ◽  
Yumi Ota ◽  
Kotaro Miyamoto ◽  
Wanaporn Yimchuen ◽  
...  
Keyword(s):  
Protein Scaffold ◽  
Small Protein

scholarly journals Fluorescent magnesium nanocomplex in a protein scaffold for cell nuclei imaging applications

RSC Advances ◽  
2015 ◽  
Vol 5 (114) ◽  
pp. 94236-94240 ◽  
Author(s):  
Alok Pandya ◽  
Apritam Tripathi ◽  
Rahul Purohit ◽  
Sanjay Singh ◽  
Manjula I. Nandasiri ◽  
...  
Keyword(s):  
Protein Complex ◽  
High Specificity ◽  
Cell Nuclei ◽  
Protein Scaffold ◽  
Photo Stability ◽  
First Time ◽  
Magnesium Nanoparticles

For the first time, we report a facile strategy for the synthesis of ultra-fine blue-green emitting fluorescent magnesium nanoparticles–protein complex (MgNC) which shows excellent photo stability and also stain cell nuclei with high specificity.

Structural and Chemical Studies of Pathological Fibers in Human Neurofibrillary Degeneration

1985 ◽  
Vol 43 ◽  
pp. 726-729
Author(s):  
K.S. Kosik ◽  
L.K. Duffy ◽  
S. Bakalis ◽  
C. Abraham ◽  
D.J. Selkoe
Keyword(s):  
Monoclonal Antibodies ◽  
Alzheimer Disease ◽  
Human Brain ◽  
Neurofibrillary Tangles ◽  
Morphological Analysis ◽  
High Specificity ◽  
Cytoskeletal Proteins ◽  
Neuritic Plaque ◽  
Protein Chemistry ◽  
Chemical Studies

The major structural lesions of the human brain during aging and in Alzheimer disease (AD) are the neurofibrillary tangles (NFT) and the senile (neuritic) plaque. Although these fibrous alterations have been recognized by light microscopists for almost a century, detailed biochemical and morphological analysis of the lesions has been undertaken only recently. Because the intraneuronal deposits in the NFT and the plaque neurites and the extraneuronal amyloid cores of the plaques have a filamentous ultrastructure, the neuronal cytoskeleton has played a prominent role in most pathogenetic hypotheses.The approach of our laboratory toward elucidating the origin of plaques and tangles in AD has been two-fold: the use of analytical protein chemistry to purify and then characterize the pathological fibers comprising the tangles and plaques, and the use of certain monoclonal antibodies to neuronal cytoskeletal proteins that, despite high specificity, cross-react with NFT and thus implicate epitopes of these proteins as constituents of the tangles.

A comparison of three cryotechniques (freeze-drying, cryosubstitution, cryosectioning) of specimen preparation for immunolabelling of bacterial antigens of Coxiella burnetii

1994 ◽  
Vol 52 ◽  
pp. 140-141
Author(s):  
T. F. McCaul ◽  
R. J. Gould
Keyword(s):  
Coxiella Burnetii ◽  
Freeze Drying ◽  
Stress Proteins ◽  
Cultured Cells ◽  
High Specificity ◽  
Specimen Preparation ◽  
Bacterial Antigens ◽  
Structural Preservation ◽  
Nuclear Ribonucleoprotein ◽  
Cellular Components

Immuno-electron microscopy has allowed the selective localisation of molecules with high resolution and high specificity. Cryopreparatory methods have provided better retention of antigenicity suitable for precise immunolabelling together with optimal structural preservation of cellular components. Cryosubstitution and cryoultramicrotomy have widely been exploited for immunolabelling. Molecular Distillation Dryer (MDD), a form of freeze-drying technique, has recently been used for immunolabelling of Plasmodium falciparum stress proteins and nuclear ribonucleoprotein particles in cultured cells. In the present study, we report the comparison of all three cryotechniques in the immunolabelling of bacterial antigens of Coxiella burnetii.The highly infectious C. burnetii was prefixed in 3% glutaraldehyde (66 mM cacodylate buffer, pH 6.8 ). The cells were then pre-embedded in 2% low-temperature agarose on Durapore hydrophilic membrane prior to cryofixation using a LifeCell CF100 metal-mirror system. A 1% glutaraldehyde in 100% methanol was used as a medium for cryosubstitution in a Reichert CS Auto Cryosubstitution apparatus.

Electronmicroscopic localization of ribonucleic acids in ciliate Bursaria truncatella during cell division

1990 ◽  
Vol 48 (3) ◽  
pp. 132-133
Author(s):  
Vladimir Popenko ◽  
Natalya Cherny ◽  
Maria Yakovleva
Keyword(s):  
Cell Division ◽  
High Specificity ◽  
Longitudinal Axis ◽  
Gold Complexes ◽  
Somatic Nucleus ◽  
Ribonucleic Acids ◽  
Biological Meaning ◽  
Bivalent Cations ◽  
Lowicryl K4m ◽  
Short Time

Highly polyploid somatic nucleus (macronucleus) of ciliate Bursaria truncatella under goes severe changes in morphology during cell division. At first, macronucleus (Ma) condences, diminishes in size and turns perpendicular to longitudinal axis of the cell. After short time, Ma turns again, elongates and only afterwards the process of division itself occurs. The biological meaning of these phenomena is not clear.Localization of RNA in the cells was performed on sections of ciliates B. truncatella, embedded in “Lowicryl K4M” at various stages: (1) before cell division (Figs. 2,3); (11) at the stage of macronucleus condensation; (111) during elongation of Ma (Fig.4); (1111) in young cells (0-5min. after division). For cytochemical labelling we used RNaseAcolloidal gold complexes (RNase-Au), which are known to bind to RNA containing cell ularstructures with high specificity. The influence of different parameters on the reliability and reproducibility of labelling was studied. In addition to the factors, discussed elsewhere, we found that the balance of mono- and bivalent cations is of great significance.

Hit and False-Alarm Rates of Selected ABR Indices in Differentiating Cochlear Disorders From Acoustic Tumors

1996 ◽  
Vol 5 (1) ◽  
pp. 90-96 ◽  
Author(s):  
Frank E. Musiek ◽  
Cynthia A. McCormick ◽  
Raymond M. Hurley
Keyword(s):  
False Alarm ◽  
Amplitude Ratio ◽  
High Specificity ◽  
Auditory Brainstem ◽  
Latency Difference ◽  
Cochlear Pathology ◽  
Brainstem Response ◽  
Absolute Latency ◽  
Two Indices ◽  
Low Sensitivity

We performed a retrospective study of 26 patients with acoustic tumors and 26 patients with otologically diagnosed cochlear pathology to determine the sensitivity (hit rate), specificity (false-alarm rate), and efficiency of six auditory brainstem response indices. In addition, a utility value was determined for each of these six indices. The I–V interwave interval, the interaural latency difference, and the absolute latency of wave V provided the highest hit rates, the best A’ values and good utility. The V/I amplitude ratio index provided high specificity but low sensitivity scores. In regard to sensitivity and specificity, using the combination of two indices provided little overall improvement over the best one-index measures.

531: Quantitative Real-Time RT-PCR for AMACR Distinguishes Prostate Cancer with High Specificity From Benign Lesions

2005 ◽  
Vol 173 (4S) ◽  
pp. 145-145 ◽  
Author(s):  
Martin Schostak ◽  
Hans Krause ◽  
Jens Köllermann ◽  
Mark Schrader ◽  
Bernd Straub ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
High Specificity ◽  
Rt Pcr ◽  
Benign Lesions
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