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Related Experiment Videos

Chemically crosslinked protein dimers: stability and denaturation effects

M P Byrne1, W E Stites

  • 1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701-1201, USA.

Protein Science : a Publication of the Protein Society
|December 1, 1995
PubMed
Summary

Staphylococcal nuclease dimers crosslinked with specific reagents showed unusual biphasic unfolding, differing from monomeric protein behavior. This suggests crosslinking alters protein denaturation pathways, particularly in the denatured state.

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Area of Science:

  • Biochemistry
  • Protein chemistry
  • Structural biology

Background:

  • Staphylococcal nuclease is a model enzyme for studying protein folding.
  • Cysteine residues are reactive sites for chemical modification and crosslinking.
  • Understanding protein unfolding mechanisms is crucial for protein engineering and drug design.

Purpose of the Study:

  • To investigate the effects of intramolecular crosslinking on the folding and unfolding thermodynamics of staphylococcal nuclease.
  • To compare the unfolding behavior of crosslinked dimeric staphylococcal nuclease with its monomeric form.
  • To explore the influence of crosslinking reagents on protein stability and denaturation pathways.

Main Methods:

  • Site-directed mutagenesis to introduce cysteine residues into staphylococcal nuclease.

Related Experiment Videos

  • Chemical crosslinking of mutant proteins using bifunctional reagents (BMH, DBP, mustard gas) and a monofunctional control (MCA).
  • Guanidine hydrochloride (GuHCl) denaturation and thermal denaturation studies to analyze protein unfolding.
  • Thermodynamic modeling (two-state and three-state models) to interpret unfolding data.

    • Main Results:

    • Crosslinking introduced cysteine residues resulted in biphasic GuHCl denaturation curves, deviating from the two-state model observed for MCA-modified proteins.
    • A three-state thermodynamic model, representing cooperative unfolding of individual subunits, best described the GuHCl denaturation of crosslinked dimers.
    • Thermal denaturation data fitted a two-state model but showed elevated van't Hoff enthalpies, suggesting altered denatured states.
    • Differences in unfolding behavior were attributed to changes in the denatured state induced by crosslinking.

    Conclusions:

    • Intramolecular crosslinking significantly alters the thermodynamic pathways of staphylococcal nuclease unfolding.
    • The denatured state of crosslinked proteins differs substantially from that of monomeric or monofunctionally modified proteins.
    • Crosslinking can stabilize proteins or introduce complexities in their folding/unfolding mechanisms, requiring advanced thermodynamic models for accurate description.