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

Enhanced Protein Thermostability by Ala --> Aib Replacement

De Filippis V1, De Antoni F, Frigo

  • 1CRIBI Biotechnology Centre, University of Padua, via Trieste 75, 35121 Padua, Italy

Biochemistry
|March 4, 1998
PubMed
Summary

Incorporating alpha-aminoisobutyric acid (Aib) into proteins enhances helical stability. Replacing alanine with Aib in thermolysin subdomain 255-316 increased melting temperatures, showing Aib can stabilize proteins.

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

Steroid-sparing effects of pentoxifylline in pulmonary sarcoidosis.

Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG·2010
Same author

Simian virus-40 sequences are a negative prognostic cofactor in patients with malignant pleural mesothelioma

Genes, chromosomes & cancer·2000
Same author

Glass transition and relaxation following PhotoPerturbation in thin polymeric films

Physical review letters·2000
Same author

Atom-selective bond breaking in a chemisorbed homonuclear molecule induced by core excitation: N2/Ru(001)

Physical review letters·2000
Same author

Nature of the short wavelength excitations in vitreous silica: An X-Ray brillouin scattering study

Physical review letters·2000
Same author

Chemical ecology of the Antarctic nudibranch Bathydoris hodgsoni Eliot, 1907: defensive role and origin of its natural products.

Journal of experimental marine biology and ecology·2000

Area of Science:

  • Protein Engineering
  • Biochemistry
  • Structural Biology

Background:

  • Alpha-aminoisobutyric acid (Aib) incorporation stabilizes short peptide helical structures by limiting backbone conformations.
  • Thermolysin's carboxy-terminal subdomain 255-316, a 62-residue fragment, forms a dimer and possesses a 3-alpha-helix structure consistent with the intact protein.

Purpose of the Study:

  • To evaluate the stabilizing effect of alpha-aminoisobutyric acid (Aib) at the protein level.
  • To investigate the conformational and stability properties of Aib-containing analogs of thermolysin's carboxy-terminal subdomain 255-316.

Main Methods:

  • Semisynthetic approach to create Aib-containing analogs of thermolysin fragment 255-316 by replacing alanine residues with Aib.
  • Circular dichroism (CD) spectroscopy (far- and near-UV) to assess secondary and tertiary structures.

Related Experiment Videos

  • Thermal unfolding monitored by ellipticity at 222 nm to determine melting temperatures (Tm).

    • Main Results:

    • Secondary and tertiary structures were fully retained in Aib-substituted analogs.
    • Melting temperatures (Tm) increased by 2.2 °C (Ala304Aib) and 5.4 °C (Ala309Aib) compared to the natural fragment (Tm = 63.5 °C).
    • A double substitution (Ala304Aib/Ala309Aib) resulted in an additive +8 °C increase in Tm, indicating significant stabilization.

    Conclusions:

    • Rational incorporation of Aib can be a general strategy to significantly stabilize proteins.
    • Stabilization effects are attributed to reduced backbone entropy and, in buried positions, hydrophobic effects.
    • Unfavorable strain energy can occur if Aib substitution is at positions with disallowed backbone angles (e.g., Ala312).