In situ Monitoring By Raman Spectroscopy of Lysozyme Conformation during “Nanotemplate” Induced Crystallization

Abstract

Using Raman spectroscopy and lysozyme, this latter as model protein, we investigate the differences in protein conformation before and after LB nanotemplate-induced crystal nucleation and growth. It was found that the main difference in lysozyme conformation is associated to the higher amount of S-S bonds in lysozyme of LB crystals, probably in C-end of protein, resulting in the higher stiffness of the lysozyme molecules and LB crystal in a whole. Growth in size of LB crystal over time is also accompanied by the formation of S-S bonds. Atomic structure, determined by X-ray diffraction, correlates Raman spectroscopy results confirm the main differences between LB and classical crystals are in terms of water molecules environment previously associated to the increased radiation stability of LB crystals.

Raman Spectroscopy of Protein Crystal Nucleation and Growth

“…The data about change in the amount of S-S bonds are in the agreement with our previous results (Nicolini et al, 2013). To relate spectral changes with the conforamtional difference of lisozyme molecules in LB and classical crystals we used following information: There are four possible disulfide bonds in lisozyme.…”

“…Assignment of peaks in lysozyme Raman spectra and their sensitivity to the invironment is shown in Table 1. Position of peaks in Raman spectra of lysozyme obtained with 532 nm laser is the same as we observed previously in lysozyme Raman spectra obtained by 632.8 nm excitation (Nicolini et al, 2013). The difference of the present study from the previous one (Nicolini et al, 2013) is that here we analyze spectral region 400-1800 cm −1 , whereas in (Nicolini et al, 2013) we focused our attention on 500-1100 cm −1 region.…”

“…Position of peaks in Raman spectra of lysozyme obtained with 532 nm laser is the same as we observed previously in lysozyme Raman spectra obtained by 632.8 nm excitation (Nicolini et al, 2013). The difference of the present study from the previous one (Nicolini et al, 2013) is that here we analyze spectral region 400-1800 cm −1 , whereas in (Nicolini et al, 2013) we focused our attention on 500-1100 cm −1 region. We should note, that in spite of the similarity of spectra in region 500-1100 cm −1 excited by 532 and 632.8 nm, there are some differences: The overall spectrum intensity at 532 nm excitation is higher than at 632.8 nm excitation, especially of Trp peaks 759 and 1008 cm −1 ; and there is no peak at 634 cm −1 attributed to S-H bond vibration in Cys residues under 532 nm excitation.…”

Protein Confinement in the Anodic Porous Alumina Microarray

“…The lysozyme Raman spectra of 532 nm laser excitation with the peaks assignment [21] was taken as the reference one ( Figure 5A), even if the frequency shift range of (400-1800 cm -1 ) is slightly different in comparison with present experiment (200-1500 cm -1 ) ( Figure 5B). Moreover, position of peaks in Raman spectra of lysozyme are similar as we observed previously in lysozyme Raman spectra obtained by 632.8 laser nm excitation [22]. The peak corresponding to acetate (487 cm -1 ) found in the obtained Raman spectra can result from the lysozyme buffer 50 mM Sodium Acetate pH 6.5.…”

“…Raman spectra of lysozyme is shown on the figure 5. We compare the Raman spectra of the lysozyme crystals, previously obtained [21,22] with the CFSE-labelled lysozyme, confined in the APA pore. Indeed, the Raman spectra peak positions of CFSE-labelled lysozyme into the APA pore are similar to those obtained for non-labelled protein in crystals (see Figure 5).…”