Thermostability of photosystem I trimers and monomers from the cyanobacterium Thermosynechococcus elongatus.

The performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermostability of photosystem I (PSI) complexes Terasaki et al. (2007), Iwuchukwu et al. (2010), Kothe et al. (2013) . To assess the thermostability of PSI complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus heating induced perturbations on the level of secondary structure of the proteins were studied. Changes were monitored by Fourier transform infrared (FT-IR) spectra in the mid-IR region upon slow heating (1°C per minute) of samples in D2O phosphate buffer (pD 7.4) from 20°C to 100°C. These spectra showed distinct changes in the Amide I region of PSI complexes as a function of the rising temperature. Absorbance at the Amide I maximum of PSI monomers (centered around 1653cm-1), gradually dropped in two temperature intervals, i.e. 60-75 and 80-90°C. In contrast, absorbance at the Amide I maximum of PSI trimers (around 1656cm-1) dropped only in one temperature interval 80-95°C. The thermal profile of the spectral shift of α-helices bands in the region 1656-1642cm-1 confirms the same two temperature intervals for PSI monomers and only one interval for trimers. Apparently, the observed absorbance changes at the Amide I maximum during heating of PSI monomers and trimers are caused by deformation and unfolding of α-helices. The absence of absorbance changes in the interval of 20-65°C in PSI trimers is probably caused by a greater stability of protein secondary structure as compared to that in monomers. Upon heating above 80°C a large part of α-helices both in trimers and monomers converts to unordered and aggregated structures. Spectral changes of PSI trimers and monomers heated up to 100°C are irreversible due to protein denaturation and non-specific aggregation of complexes leading to new absorption bands at 1618-1620cm-1. We propose that monomers shield the denaturation sensitive sides at the monomer/monomer interface within a trimer, making the oligomeric structure more stable against thermal stress.

Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: watching a protease eat a protein.

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of ß-lactoglobulin (ß-LG) and ß-casein (ß-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded ß-LG show a decrease around 1634 cm(-1), suggesting a decrease of ß-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm(-1) band decreases for ß-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ∼1594 cm(-1) and ∼1406 cm(-1) increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for ß-LG and ß-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.