Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Probing beta-lactamase structure and function using random replacement mutagenesis.

T Palzkill1, D Botstein

  • 1Department of Genetics, School of Medicine, Stanford University, California 94305.

Proteins
|September 1, 1992
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Identification and characterization of beta-lactamase inhibitor protein-II (BLIP-II) interactions with beta-lactamases using phage display.

Protein engineering, design & selection : PEDS·2010
Same author

Editorial.

Molecular biology of the cell·2006
Same author

Genomic perspective and cancer.

Cold Spring Harbor symposia on quantitative biology·2004
Same author

The Gene Ontology (GO) database and informatics resource.

Nucleic acids research·2003
Same author

Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae.

Cold Spring Harbor symposia on quantitative biology·2003
Same author

Genome-wide scan of bipolar disorder in 65 pedigrees: supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12.

Molecular psychiatry·2003
Same journal

Engineered HSP90-MP65 Bivalent Fusion Antigen: A Novel Vaccine Candidate Against Invasive Candidiasis.

Proteins·2026
Same journal

Physics-Based Energy Functions for Computational Protein Design.

Proteins·2026
Same journal

Impact of Stabilizing Osmolytes on the Conformational Dynamics of Human and Rat Islet Amyloid Polypeptides.

Proteins·2026
Same journal

Stabilization of Bone Morphogenetic Protein-2 at Physiological pH: Contrasting Roles of CHAPS and Arginine in Aggregation Inhibition.

Proteins·2026
Same journal

Structural Insights Into the Function of Leishmania major Adenylosuccinate Lyase.

Proteins·2026
Same journal

Generalizing the Gaussian Network Model: Spanning-Tree Thermodynamics Shows Entropy-Driven KRAS Activation.

Proteins·2026
See all related articles

A new mutagenesis technique reveals that most random DNA changes in TEM-1 beta-lactamase still yield functional proteins. Critical protein regions, identified by this method, are often near the active site or in the hydrophobic core.

Area of Science:

  • Protein engineering and structural biology
  • Molecular genetics and mutagenesis
  • Enzyme function and evolution

Background:

  • Understanding protein structure-function relationships is crucial for protein engineering.
  • Analytical mutagenesis techniques help identify critical amino acid residues.
  • TEM-1 beta-lactamase is a well-studied enzyme relevant to antibiotic resistance.

Purpose of the Study:

  • To develop and apply a novel analytical mutagenesis technique to assess protein tolerance to sequence variation.
  • To identify regions within TEM-1 beta-lactamase that are critical for its structure and function.
  • To compare sequence requirements with evolutionary conservation in related beta-lactamases.

Main Methods:

  • Randomizing short DNA stretches (3-6 codons) within a target gene.

Related Experiment Videos

  • Quantifying the percentage of functional proteins from libraries of random sequence substitutions.
  • Applying the method across multiple regions of the TEM-1 beta-lactamase gene.
  • Analyzing X-ray structures and comparing sequence conservation among homologous beta-lactamases.

    • Main Results:

    • TEM-1 beta-lactamase demonstrates high tolerance to amino acid substitutions, with an average of 44% of mutants retaining function.
    • A significant proportion (20%) of random substitutions resulted in functional enzyme expression, secretion, and folding.
    • Specific regions, particularly those near the active site or in the hydrophobic core, were found to be highly sensitive to sequence changes.
    • Functional sequence analysis provided detailed insights into amino acid requirements for specific regions.

    Conclusions:

    • The developed analytical mutagenesis technique provides a global view of protein sequence criticality.
    • TEM-1 beta-lactamase is remarkably robust, with most mutations not abolishing function.
    • Sensitive regions identified are consistent with structural and functional importance, particularly active site and core residues.
    • The findings contribute to understanding protein evolution and designing enzymes with altered properties.