Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles.
In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.
Majumder
Parijat
P
C.K. Majumdar Laboratory, S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India. lgahu@163.com
Sarkar
Rupa
R
Shaw
Ajay Kumar
AK
Chakraborty
Anjan
A
Pal
Samir Kumar
SK
D000327:Adsorption; D002918:Chymotrypsin / Q000737:chemistry*; D003374:Coumarins / Q000737:chemistry; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry; D004789:Enzyme Activation; D004996:Ethidium / Q000737:chemistry; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008823:Micelles; D015394:Molecular Structure; D049329:Nanostructures / Q000737:chemistry*; D010316:Particle Size; D011744:Pyrimidinones / Q000737:chemistry; D020112:Rhodamine 123 / Q000737:chemistry; D012995:Solubility; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013997:Time Factors
C514684:7-(ethylamino)-4-trifluoromethylcoumarin; D003374:Coumarins; D008823:Micelles; D011744:Pyrimidinones; D004143:Dioctyl Sulfosuccinic Acid; D020112:Rhodamine 123; C003954:merocyanine dye; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D004996:Ethidium