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Related Concept Videos

Molecular Chaperones and Protein Folding03:00

Molecular Chaperones and Protein Folding

The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
Molecular Chaperones and Protein Folding03:00

Molecular Chaperones and Protein Folding

The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Protein Folding01:25

Protein Folding

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...

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Related Experiment Video

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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions
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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

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Conversion of scFv peptide-binding specificity for crystal chaperone development.

Jennifer C Pai1, Jeffrey A Culver, Jason E Drury

  • 1Department of Chemical Engineering, University of Texas at Austin, TX 78712, USA.

Protein Engineering, Design & Selection : PEDS
|January 11, 2011
PubMed
Summary

Researchers engineered new single-chain variable fragments (scFvs) that act as chaperones for protein crystallization. These engineered scFvs can bind specific peptide tags, aiding structure determination for recalcitrant proteins.

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Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
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Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Published on: December 30, 2016

Area of Science:

  • Structural Biology
  • Protein Engineering
  • Biochemistry

Background:

  • Many proteins are difficult to crystallize for X-ray crystallography, hindering structure determination.
  • Co-crystallization with antibody fragments is an emerging strategy, especially for membrane proteins.
  • Existing antibody fragments may lack desired specificity or expression characteristics.

Purpose of the Study:

  • To develop novel recombinant single-chain variable fragments (scFvs) with engineered binding specificities.
  • To create 'chaperone' scFvs that facilitate the crystallization of challenging proteins.
  • To explore new strategies for improving protein structure determination.

Main Methods:

  • Modification of a known hexa-histidine-specific scFv (3D5) via complementary determining region alteration and random mutagenesis.
  • Application of phage display to select scFv variants with altered binding properties.
  • Co-crystallization studies, gel filtration, and X-ray crystallography to characterize engineered scFvs and their complexes.

Main Results:

  • Engineered scFvs demonstrated enhanced expression, solubility (up to 16.6 mg/ml), and sub-micromolar affinity for hexa-histidine tags.
  • New scFv variants with nanomolar affinity for the EE hexa-peptide (EYMPME) were successfully generated.
  • The crystal structure of an EE-binding scFv revealed a complementary binding surface and a channel, distinct from the parent scFv's crystal packing.

Conclusions:

  • Engineered scFvs serve as novel protein chaperones, binding specific peptide tags to aid crystallization.
  • These 'chaperone' scFvs offer a versatile platform for structure determination of recalcitrant proteins.
  • This approach may reduce the need for extensive screening of antibody libraries for crystallization partners.