Engineering of a sulfotyrosine-recognizing small protein scaffold for the study of protein tyrosine O-sulfation

2019 ◽  
pp. 67-89 ◽  
Author(s):  
Justin Lawrie ◽  
Wei Niu ◽  
Jiantao Guo
Keyword(s):  
Protein Scaffold ◽  
Small Protein ◽  
Protein Tyrosine

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

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

Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
Author(s):  
Karim Rezaul ◽  
Shigeru Yanagi ◽  
Kiyonao Sada ◽  
Takanobu Taniguchi ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Platelet Activating Factor ◽  
Kinase Assay ◽  
Potential Candidate ◽  
Protein Tyrosine ◽  
Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

Naturally Occurring Homoisoflavonoids as Potent Protein Tyrosine Kinases Inhibitors

Planta Medica ◽  
2008 ◽  
Vol 74 (03) ◽  
Author(s):  
Y Ye ◽  
LG Lin ◽  
H Xie ◽  
HL Li ◽  
HL Jiang ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Naturally Occurring

1742-P: Targeting the Low Molecular Weight Protein Tyrosine Phosphatase for Obesity-Associated Diabetes Therapy

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 1742-P
Author(s):  
STEPHANIE M. STANFORD ◽  
MICHAEL A. DIAZ ◽  
JIWEN J. ZOU ◽  
ROBERT J. ARDECKY ◽  
ANTHONY PINKERTON ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Low Molecular Weight ◽  
Molecular Weight Protein ◽  
Diabetes Therapy ◽  
Protein Tyrosine ◽  
Weight Protein ◽  
Low Molecular Weight Protein
Sign in / Sign up

Export Citation Format

Share Document