A Holistic Approach to Protein Secondary Structure Characterization Using Amide I Band Raman Spectroscopy

Sane, Samir U.; Cramer, Steven M.; Przybycien, Todd M.

doi:10.1006/abio.1999.4034

Cited by 110 publications

(134 citation statements)

References 44 publications

(59 reference statements)

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“…15 In addition, the protein secondary structure can be characterized by analyzing the protein amide I Raman band. 17 Thus, it is possible to simultaneously follow the reduction of the disulfide bridges and the related protein unfolding, which is revealed by the changes in the protein secondary structure. A Raman spectroscopy study of the disulfide bridge exchange reaction between serum albumins and DTT in aqueous solutions is presented in this article.…”

Section: Introductionmentioning

confidence: 99%

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

et al. 2008

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The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.

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Section: Introductionmentioning

confidence: 99%

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

et al. 2008

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“…Broadening of the Am I peak is greatest in the spectrum of the intact virus. On the lower-energy side of the Am I vibration, the 1,572 cm Ϫ1 band, which originates from ring breathing modes, both from Trp as well as from adenine and guanine of RNA (39,41), shows higher intensity in the heated particles, especially in the spectra of the particles treated at 60°C.…”

Section: Resultsmentioning

confidence: 98%

“…On many occasions, the scattering intensity of viruses is small, and the changes at low concentrations are hidden inside the overruling background spectra. This leads to challenges in detecting the Raman signal of the sample and accurately subtracting the solvent and other background from the weak viral spectra (41). It is also important to keep in mind that some buffers include groups that have vibrations similar to those of many biological samples or, at worst, are quite reactive (51) and therefore can, if not obscure the spectra, at least skew the interpretation.…”

Section: Discussionmentioning

confidence: 99%

“…Care was taken to remove the contribution of the wagging mode of water molecules from the Raman data of the diluted samples, which has been determined to be essential for a proper analysis of the Am I modes (41). The Raman Am I band centered at 1,668 cm Ϫ1 indicates ␤-sheets as the predominant secondary structure of the capsid, which is consistent with the known structure of the major capsid proteins, VP1, VP2, and VP3, in EV1 (58).…”

Section: Discussionmentioning

confidence: 99%

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Raman Spectroscopic Signatures of Echovirus 1 Uncoating

Ruokola

Dadu

Kazmertsuk

et al. 2014

J Virol

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In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact virion, those of the proteins of the heat-treated particles indicated reduced ␣-helix content with respect to ␤-sheets and coil structures. Changes observed in tryptophan and tyrosine signals suggest an increasingly hydrophilic environment around these residues. RNA signals revealed a change in the environment of the genome and in its conformation. The ionized-carbonyl vibrations showed small changes between the intact virion and the uncoating intermediate, which points to cleavage of salt bridges in the protein structure during the uncoating process. In conclusion, our data reveal distinguishable Raman signatures of the intact, intermediate, and disrupted EV1 particles. These changes indicate structural, chemical, and solute-solvent alterations in the genome and in the capsid proteins and lay the essential groundwork for investigating the uncoating of EV1 and related viruses in real time. IMPORTANCEIn order to combat virus infection, we need to know the details of virus uncoating. We present here the novel Raman signatures for opened and intact echovirus 1. This gives hope that the signatures may be used in the near future to evaluate the ambient conditions in endosomes leading to virus uncoating using, e.g., coherent anti-Stokes Raman spectroscopy (CARS) imaging. These studies will complement structural studies on virus uncoating. In addition, Raman/CARS imaging offers the possibility of making dynamic live measurements in vitro and in cells which are impossible to measure by, for example, cryo-electron tomography. Furthermore, as viral Raman spectra can be overwhelmed with various contaminants, our study is highly relevant in demonstrating the importance of sample preparation for Raman spectroscopy in the field of virology. Picornaviruses are small, nonenveloped human pathogens able to cause a wide range of illnesses (1). The subgroup enteroviruses include clinically important echoviruses, coxsackieviruses, and poliovirus. These viruses cause a variety of diseases varying from mild infections to aseptic meningitis, heart muscle damage, and paralysis. Enteroviruses have also recently been associated with chronic diseases such as type 1 diabetes, cardiomyopathies, and atherosclerosis (2, 3). Structural details of these viruses have been obtained by means of X-ray crystallography or at a lower resolution by cryo-electron microsc...

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“…Bands at 1640 and 1660 cm −1 are assigned to α-helix protein secondary structure while the band at 1680 cm −1 is assigned to β-sheet protein secondary structure [29][30][31]. Muscle is composed largely of actin and myosin, both which are more alpha helical in structure than collagen [32].…”

Section: Discussionmentioning

confidence: 99%