Raman spectroscopy of protein crystal nucleation and growth

Using Raman spectroscopy and the lysozyme as model system, we investigate the differences in protein conformation before and after Langmuir- Blodgett 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 to the above pointing to the main differences between LB classical crystals in terms of water molecules environment previously associated to the increased radiation stability of LB crystals

Raman Scattering:From Structural Biology to Medical Applications

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed

SERS and Raman spectra of WT and mutant cytochromes c

Example Raman and SERS spectra of WT cytochrome c and three different mutants: p { margin-bottom: 0.1in; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 10pt; }p.ctl { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }a.western:link { }a.ctl:link { } <p><b>Mutant 1</b> T78N/K79Y/M80I/I81M/F82N<br> p { margin-bottom: 0.1in; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 10pt; }p.ctl { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }a.western:link { }a.ctl:link { } </p><p><b>Mutant 2</b> T78S/K79P<br> p { margin-bottom: 0.1in; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 10pt; }p.ctl { font-family: "NanumGothic","Times New Roman"; font-size: 10pt; }a.western:link { }a.ctl:link { } </p><p><b>Mutant 3</b> I81Y/A83Y/G84N</p><p>"2ag_3mutant...." in the file name means that this is SERS spectrum of the mutant (1,2 or 3 or WT) cytochrome.<br></p><p>"mutant..sdt..." or "WT.._sdt.." in the file name means that this is Raman spectrum of the mutant or WT cytochrome c treated with sodium dithionite<br></p

Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman microspectroscopy

This paper presents a nonivasive approach to study redox state of reduced cytochromes [Image: see text], [Image: see text] and [Image: see text] of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes [Image: see text], [Image: see text] and [Image: see text]. The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes [Image: see text], [Image: see text] and [Image: see text] in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes [Image: see text], [Image: see text] and [Image: see text] in the rod-shaped cardiomyocytes caused by H(2)O(2)-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome [Image: see text] in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data

In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions

Unraveling cell processes: interference imaging interwoven with data analysis

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1–0.6 Hz) result from plasma membrane processes and that higher frequency variations (20–26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties