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Related Concept Videos

Protein Folding01:22

Protein Folding

Overview
Protein Folding01:22

Protein Folding

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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...
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 Organization01:24

Protein Organization

Proteins are polymers of amino acid residues. They are versatile and responsible for different cellular functions, including DNA replication, molecular transport, catalysis, and structural support. Proteins have a hierarchical structure comprising at least three levels of organization: primary, secondary, and tertiary structure. Some large proteins have a quaternary structure where individual protein subunits are linked together.
The primary structure of a protein is its amino acid sequence.

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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
14:25

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Published on: December 12, 2017

Selecting folded proteins from a library of secondary structural elements.

James J Graziano1, Wenshe Liu, Roshan Perera

  • 1Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of the American Chemical Society
|December 11, 2007
PubMed
Summary

Researchers developed a novel protein evolution strategy to create new polypeptides. This method successfully generated soluble proteins with defined secondary structures, some with no known homology.

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Area of Science:

  • Protein Engineering
  • Synthetic Biology
  • Biochemistry

Background:

  • Designing novel proteins with specific structures and functions is a key challenge in protein engineering.
  • Understanding the relationship between sequence, structure, and stability is crucial for de novo protein design.

Purpose of the Study:

  • To develop and validate a protein evolution strategy for generating novel polypeptides with defined secondary structures.
  • To create a library of new protein sequences and identify soluble, stable variants in vivo.

Main Methods:

  • Assembling double-stranded DNA fragments encoding Escherichia coli secondary structural elements (alpha-helices, beta-strands, loops) into semirandom sequences.
  • Inserting generated polypeptide libraries into an enhanced green fluorescent protein (EGFP) fusion vector.
  • Screening library members using fluorescence-activated cell sorting (FACS) and characterizing soluble clones via digital PCR (dPCR).

Main Results:

  • A library of novel polypeptides was generated and screened, yielding 1149 high-fluorescence colonies from approximately 10^8 clones.
  • Four soluble clones with varying secondary structures were identified, including one homologous to marine aspartate racemase and others with no known sequence homology.
  • Selected novel polypeptides exhibited significant alpha-helical content, reversible folding, and pH-sensitive binding, indicating stable, structured proteins.

Conclusions:

  • The described protein evolution strategy is effective in generating novel polypeptide sequences with predictable secondary structures.
  • This approach can yield soluble and stable proteins, including those with no homology to existing protein databases.
  • The identified novel polypeptides represent promising candidates for further functional characterization and biotechnological applications.