Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Abstract

Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture). We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm−1 to 1632 cm−1, indicating a shift from α‐helical structure to β‐sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β‐sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

“…Widely used methods for probing such effects entail measuring changes in infrared absorbance, circular dichroism, or intrinsic fluorescence after exposure of the interface to protein solution (2,(31)(32)(33)(34)(35)(36). These methods can detect shifts in the average secondary structure of the adsorbed protein layer over time without a priori knowledge of protein structure.…”

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Changes in the protein's relative composition in secondary structure and also in the tertiary fold on immobilization can be monitored using FTIR, as shown in Table 3. Protein surface interactions, hydrogen bond formations and other bond parameters are detectable by FTIR too [21,[93][94][95]. FTIR studies of different enzymes revealed retention of secondary structural composition in the immobilizate.…”

“…With other words, more convenient materials (e.g. mesoporous silica, porous polymer beads) using FTIR, the interference with the measurement is unfortunately quite significant [22,38,41,94,[104][105][106][107].…”

Experimental test of absorption flattening correction for circular dichroism of particle suspensions

“…We have recently shown that it is possible to make useful CD measurements on such particles. 7,8 These made use of a rotating sample cell to keep particles in suspension, and placement close to the detector to minimize scattering effects. All CD measurements on particle suspensions may be affected by the artifact of absorption flattening.…”