Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.
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.
Medintz
Igor L
IL
Center for Bio/Molecular Science and Engineering, Code 6900, US Naval Research Laboratory, Washington, DC 20375, USA. imedintz@cbmse.nrl.navy.mil
Clapp
Aaron R
AR
Brunel
Florence M
FM
Tiefenbrunn
Theresa
T
Uyeda
H Tetsuo
HT
Chang
Eddie L
EL
Deschamps
Jeffrey R
JR
Dawson
Philip E
PE
Mattoussi
Hedi
H
D000595:Amino Acid Sequence; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D049329:Nanostructures / Q000737:chemistry; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis; D045663:Quantum Dots
D010455:Peptides; D011480:Protease Inhibitors; D010447:Peptide Hydrolases