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Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure
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Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure

A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: closed (31 July 2021) | Viewed by 26619

Special Issue Editors

Prof. Dr. Paola Taddei

E-Mail Website
Guest Editor
Department of Biomedical and Neuromotor Sciences, University of Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy
Interests: vibrational spectroscopy; apatite; mineralization; protein conformation; silk fibroin; biomaterials
Dr. Michele Di Foggia

E-Mail Website
Guest Editor
Department of Biomedical and Neuromotor Sciences, University of Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy
Interests: protein secondary and tertiary structure; protein interaction with metal ions; biomimetic coatings for biomedical devices; vibrational spectroscopy; SERS; tissue engineering; silk fibroin
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

It is a great pleasure to announce this Special Issue on “Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure”.

Protein structure has been investigated for more than four decades by means of vibrational spectroscopy: the specific band assignments (i.e., marker bands of amino acid side chains, disulfide bridge conformation, metal coordination) and signatures of the secondary structure have been established. The assignments are usually based on model compounds (e.g., amino acids or short peptides) and may be universal for the whole class of molecules (i.e., amide bands of proteins are assigned to polypeptide backbone modes).

Moreover, information on the presence of some functional moieties (such as –SH, S–S, etc.), the microenvironment of some amino acid residues (such as Tyr), the strength of hydrogen bonding, and the interaction between charged side chains (i.e., NH3+, COO−) or between metal ions and amino acids can be obtained.

It is well known that protein function is directly related to the resulting 3D structure and, therefore, vibrational techniques were used to monitor structural changes in many different situations:

    -  After adsorption on a surface (i.e., the case of the biomimetic approach on biomaterials surface), studying also the adsorbed–adsorbent interactions (covalent, electrostatic, chemical bonds formation, hydrogen and van der Waals bonds, hydrophobic and hydrophilic interactions);

    -  In denaturing conditions (i.e. after heating and/or pH changes);

    -  After interaction with other molecules (biomolecules as cellular membranes or grafting agents that can modify the properties of materials of proteinaceous origin);

    -  In physiological processes (i.e. the mineralization of collagen fibers, under free radical stress).

The study of metal-to-protein binding sites has been widely carried out by means of Raman resonance conditions and, in many cases, decades before the determination of protein structure by X-rays or NMR techniques.

More recently, surface-enhanced Raman scattering (SERS) on noble metal colloids has been successfully applied to the study of proteins in aqueous systems at very low concentrations (thus, similar to the physiological environment).

Prof. Dr. Paola Taddei
Dr. Michele Di Foggia
Guest Editors

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Keywords

  • Raman spectroscopy
  • IR spectroscopy
  • ATR
  • imaging
  • SERS
  • microspectroscopy
  • secondary structure
  • hydrogen bond
  • disulfide bridges
  • amino acids
  • side chains
  • adsorption on surface
  • structure–function relationship
  • metal–binding proteins

Published Papers (9 papers)

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Research

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23 pages, 19188 KiB  
Article
Vibrational Study on Structure and Bioactivity of Protein Fibers Grafted with Phosphorylated Methacrylates
by Michele Di Foggia, Masuhiro Tsukada and Paola Taddei
Molecules 2021, 26(21), 6487; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26216487 - 27 Oct 2021
Cited by 1 | Viewed by 1814
Abstract
In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted [...] Read more.
In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted with phosmer CL and phosmer M (commercial names, i.e., methacrylate monomers containing phosphate groups in the molecular side chain) with different weight gains. Both phosmers were recently proposed as flame retarding agents, and their chemical composition suggested a possible application in bone tissue engineering. IR and Raman spectroscopy were used to disclose the possible structural changes induced by grafting and identify the most reactive amino acids towards the phosmers. The same techniques were used to investigate the nucleation of a calcium phosphate phase on the surface of the samples (i.e., bioactivity) after ageing in simulated body fluid (SBF). The phosmers were found to polymerize onto the biopolymers efficiently, and tyrosine and serine underwent phosphorylation (monitored through the strengthening of the Raman band at 1600 cm−1 and the weakening of the Raman band at 1400 cm−1, respectively). In grafted wool keratin, cysteic acid and other oxidation products of disulphide bridges were detected together with sulphated residues. Only slight conformational changes were observed upon grafting, generally towards an enrichment in ordered domains, suggesting that the amorphous regions were more prone to react (and, sometimes, degrade). All samples were shown to be bioactive, with a weight gain of up to 8%. The most bioactive samples contained the highest phosmers amounts, i.e., the highest amounts of phosphate nucleating sites. The sulphate/sulphonate groups present in grafted wool samples appeared to increase bioactivity, as shown by the five-fold increase of the IR phosphate band at 1040 cm−1. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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18 pages, 1849 KiB  
Article
FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins
by Iwona Stawoska, Aleksandra Wesełucha-Birczyńska, Andrzej Skoczowski, Michał Dziurka and Jacek Waga
Molecules 2021, 26(17), 5388; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26175388 - 04 Sep 2021
Cited by 13 | Viewed by 2708
Abstract
Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions [...] Read more.
Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αβγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl− (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in β-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl− line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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21 pages, 1836 KiB  
Article
Tracking the Amide I and αCOO− Terminal ν(C=O) Raman Bands in a Family of l-Glutamic Acid-Containing Peptide Fragments: A Raman and DFT Study
by Ashley E. Williams, Nathan I. Hammer, Ryan C. Fortenberry and Dana N. Reinemann
Molecules 2021, 26(16), 4790; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26164790 - 07 Aug 2021
Cited by 4 | Viewed by 2032
Abstract
The E-hook of β-tubulin plays instrumental roles in cytoskeletal regulation and function. The last six C-terminal residues of the βII isotype, a peptide of amino acid sequence EGEDEA, extend from the microtubule surface and have eluded characterization with classic X-ray crystallographic techniques. The [...] Read more.
The E-hook of β-tubulin plays instrumental roles in cytoskeletal regulation and function. The last six C-terminal residues of the βII isotype, a peptide of amino acid sequence EGEDEA, extend from the microtubule surface and have eluded characterization with classic X-ray crystallographic techniques. The band position of the characteristic amide I vibration of small peptide fragments is heavily dependent on the length of the peptide chain, the extent of intramolecular hydrogen bonding, and the overall polarity of the fragment. The dependence of the E residue’s amide I ν(C=O) and the αCOO− terminal ν(C=O) bands on the neighboring side chain, the length of the peptide fragment, and the extent of intramolecular hydrogen bonding in the structure are investigated here via the EGEDEA peptide. The hexapeptide is broken down into fragments increasing in size from dipeptides to hexapeptides, including EG, ED, EA, EGE, EDE, DEA, EGED, EDEA, EGEDE, GEDEA, and, finally, EGEDEA, which are investigated with experimental Raman spectroscopy and density functional theory (DFT) computations to model the zwitterionic crystalline solids (in vacuo). The molecular geometries and Boltzmann sum of the simulated Raman spectra for a set of energetic minima corresponding to each peptide fragment are computed with full geometry optimizations and corresponding harmonic vibrational frequency computations at the B3LYP/6-311++G(2df,2pd) level of theory. In absence of the crystal structure, geometry sampling is performed to approximate solid phase behavior. Natural bond order (NBO) analyses are performed on each energetic minimum to quantify the magnitude of the intramolecular hydrogen bonds. The extent of the intramolecular charge transfer is dependent on the overall polarity of the fragment considered, with larger and more polar fragments exhibiting the greatest extent of intramolecular charge transfer. A steady blue shift arises when considering the amide I band position moving linearly from ED to EDE to EDEA to GEDEA and, finally, to EGEDEA. However, little variation is observed in the αCOO− ν(C=O) band position in this family of fragments. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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15 pages, 5388 KiB  
Article
On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy
by Jelica Milošević, Radivoje Prodanović and Natalija Polović
Molecules 2021, 26(4), 970; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26040970 - 12 Feb 2021
Cited by 20 | Viewed by 2781
Abstract
Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, [...] Read more.
Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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19 pages, 3713 KiB  
Article
Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus
by Ephrahime S. Traore, Jiasong Li, Tapiwa Chiura, Jiafeng Geng, Ankita J. Sachla, Francis Yoshimoto, Zehava Eichenbaum, Ian Davis, Piotr J. Mak and Aimin Liu
Molecules 2021, 26(3), 549; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26030549 - 21 Jan 2021
Cited by 6 | Viewed by 2654
Abstract
HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein [...] Read more.
HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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11 pages, 935 KiB  
Communication
Far-Off Resonance: Multiwavelength Raman Spectroscopy Probing Amide Bands of Amyloid-β-(37–42) Peptide
by Martynas Talaikis, Simona Strazdaitė, Mantas Žiaunys and Gediminas Niaura
Molecules 2020, 25(15), 3556; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25153556 - 04 Aug 2020
Cited by 10 | Viewed by 3271
Abstract
Several neurodegenerative diseases, like Alzheimer’s and Parkinson’s are linked with protein aggregation into amyloid fibrils. Conformational changes of native protein into the β-sheet structure are associated with a significant change in the vibrational spectrum. This is especially true for amide bands which are [...] Read more.
Several neurodegenerative diseases, like Alzheimer’s and Parkinson’s are linked with protein aggregation into amyloid fibrils. Conformational changes of native protein into the β-sheet structure are associated with a significant change in the vibrational spectrum. This is especially true for amide bands which are inherently sensitive to the secondary structure of a protein. Raman amide bands are greatly intensified under resonance conditions, in the UV spectral range, allowing for the selective probing of the peptide backbone. In this work, we examine parallel β-sheet forming GGVVIA, the C-terminus segment of amyloid-β peptide, using UV–Vis, FTIR, and multiwavelength Raman spectroscopy. We find that amide bands are enhanced far from the expected UV range, i.e., at 442 nm. A reasonable two-fold relative intensity increase is observed for amide II mode (normalized according to the δCH2/δCH3 vibration) while comparing 442 and 633 nm excitations; an increase in relative intensity of other amide bands was also visible. The observed relative intensification of amide II, amide S, and amide III modes in the Raman spectrum recorded at 442 nm comparing with longer wavelength (633/785/830 nm) excited spectra allows unambiguous identification of amide bands in the complex Raman spectra of peptides and proteins containing the β-sheet structure. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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Review

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24 pages, 2576 KiB  
Review
Two-dimensional Infrared Spectroscopy Reveals Better Insights of Structure and Dynamics of Protein
by Kiran Sankar Maiti
Molecules 2021, 26(22), 6893; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26226893 - 16 Nov 2021
Cited by 5 | Viewed by 2097
Abstract
Proteins play an important role in biological and biochemical processes taking place in the living system. To uncover these fundamental processes of the living system, it is an absolutely necessary task to understand the structure and dynamics of the protein. Vibrational spectroscopy is [...] Read more.
Proteins play an important role in biological and biochemical processes taking place in the living system. To uncover these fundamental processes of the living system, it is an absolutely necessary task to understand the structure and dynamics of the protein. Vibrational spectroscopy is an established tool to explore protein structure and dynamics. In particular, two-dimensional infrared (2DIR) spectroscopy has already proven its versatility to explore the protein structure and its ultrafast dynamics, and it has essentially unprecedented time resolutions to observe the vibrational dynamics of the protein. Providing several examples from our theoretical and experimental efforts, it is established here that two-dimensional vibrational spectroscopy provides exceptionally more information than one-dimensional vibrational spectroscopy. The structural information of the protein is encoded in the position, shape, and strength of the peak in 2DIR spectra. The time evolution of the 2DIR spectra allows for the visualisation of molecular motions. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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19 pages, 2660 KiB  
Review
Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies
by Célia M. Silveira, Lidia Zuccarello, Catarina Barbosa, Giorgio Caserta, Ingo Zebger, Peter Hildebrandt and Smilja Todorovic
Molecules 2021, 26(16), 4852; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26164852 - 11 Aug 2021
Cited by 1 | Viewed by 2463
Abstract
Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal [...] Read more.
Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN− ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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21 pages, 370 KiB  
Review
Effects of Physical and Chemical Factors on the Structure of Gluten, Gliadins and Glutenins as Studied with Spectroscopic Methods
by Konrad Kłosok, Renata Welc, Emilia Fornal and Agnieszka Nawrocka
Molecules 2021, 26(2), 508; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26020508 - 19 Jan 2021
Cited by 33 | Viewed by 5657
Abstract
This review presents applications of spectroscopic methods, infrared and Raman spectroscopies in the studies of the structure of gluten network and gluten proteins (gliadins and glutenins). Both methods provide complimentary information on the secondary and tertiary structure of the proteins including analysis of [...] Read more.
This review presents applications of spectroscopic methods, infrared and Raman spectroscopies in the studies of the structure of gluten network and gluten proteins (gliadins and glutenins). Both methods provide complimentary information on the secondary and tertiary structure of the proteins including analysis of amide I and III bands, conformation of disulphide bridges, behaviour of tyrosine and tryptophan residues, and water populations. Changes in the gluten structure can be studied as an effect of dough mixing in different conditions (e.g., hydration level, temperature), dough freezing and frozen storage as well as addition of different compounds to the dough (e.g., dough improvers, dietary fibre preparations, polysaccharides and polyphenols). Additionally, effect of above mentioned factors can be determined in a common wheat dough, model dough (prepared from reconstituted flour containing only wheat starch and wheat gluten), gluten dough (lack of starch), and in gliadins and glutenins. The samples were studied in the hydrated state, in the form of powder, film or in solution. Analysis of the studies presented in this review indicates that an adequate amount of water is a critical factor affecting gluten structure. Full article
(This article belongs to the Special Issue Vibrational Spectroscopy as a Versatile Tool To Predict Protein Structure)
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