Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates

Nat Mater. 2006 Jul;5(7):581-9. doi: 10.1038/nmat1676. Epub 2006 Jun 25.

Abstract

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Computer Simulation
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods*
  • Molecular Sequence Data
  • Nanostructures / chemistry
  • Peptide Hydrolases / analysis*
  • Peptide Hydrolases / metabolism
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protease Inhibitors / analysis
  • Quantum Dots*

Substances

  • Peptides
  • Protease Inhibitors
  • Peptide Hydrolases