Raman Spectroscopy of Proteins

Benevides, James M.; Overman, Stacy A.; Thomas, George J.

doi:10.1002/0471140864.ps1708s33

Cited by 47 publications

(33 citation statements)

References 125 publications

(276 reference statements)

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“…Amide I, this is primarily characterized by carbonyl stretch at 1630–1670 cm −1 and intense. Thus, α‐helix is characterized by 1645–1660 cm −1 , β‐sheet at 1665–1680 cm −1 and random coil at 1660–1670 cm −1 are intense in both IR and Raman . However, the presence of amide II at frequency 1530–1550 cm −1 and amide III (1230 cm −1 ), Raman‐active occur at 1230–1300 cm −1 .…”

Section: Discussionmentioning

confidence: 99%

Thermostable lipase fromGeobacillussp. Iso5: Bioseparation, characterization and native structural studies

Mahadevan

Neelagund

2013

J. Basic Microbiol.

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The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was purified to homogeneity by ultrafiltration, 6% cross-linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7-fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS-PAGE and MALDI-TOF MS/MS spectroscopy. The purified enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70 °C and about 35% activity at 85 °C for 2 h. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca(2+) and Mg(2+) and strongly inhibited by HgCl2 , PMSF, DTT, K(+) , Co(2+) , and Zn (2+) . EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α-helix and 64% β-sheet which was determined by Circular dichromism, FTIR, and Raman Spectroscopy.

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Section: Discussionmentioning

confidence: 99%

Thermostable lipase fromGeobacillussp. Iso5: Bioseparation, characterization and native structural studies

Mahadevan

Neelagund

2013

J. Basic Microbiol.

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“…Furthermore, because of their conjugated character, the aromatic rings are able to strongly modulate the electronic polarizability, thus providing medium‐to‐intense characteristic Raman bands. Detailed reviews on Raman spectroscopy of proteins report on the use of a number of these Raman lines located in the 1650–600 cm −1 spectral region, in probing the hydrophobic/hydrophilic environment of the peptide chains, as well as on their interactions with metal ions …”

Section: Introductionmentioning

confidence: 99%

Characteristic Raman lines of phenylalanine analyzed by a multiconformational approach

Hernández

Pflüger

Kruglik

et al. 2013

J Raman Spectroscopy

129

106

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Thanks to a considerable modulation of electronic polarizability, six phenylalanine (Phe) vibration modes located at ca. 1606, 1586, 1207, 1031, 1004 and 622 cm−1 appear as intense or medium bands in the Raman spectra of peptides and proteins, as confirmed by the Raman data collected from free amino acid, somatostatin and bovine serum albumin (BSA). To get information on the nature and location of these lines, we resorted to a multiconformational analysis which consists in a systematic investigation of the structural and vibrational features of hydrated Phe in a conformational space depending on four angular variables: φ, ψ, χ1 and χ2. The first two variables correspond to the Phe backbone torsion angles, whereas the latter two refer to its side chain. Based on a protocol described in an accompanying report on glycine and its protonated and deprotonated species, we have prepared an initial set of 123 initial clusters of Phe + 5H2O, including all plausible values of the above mentioned conformational angles. The results of their geometry optimization, by means of the density functional theory using the B3LYP hybrid functional, were first analyzed through the comparison between the E(χ1, χ2) energy maps obtained either by an explicit or by an implicit hydration model. Then, a set of nine doubly minimized clusters corresponding to the deepest local minima were used for further structural and vibrational analysis. Beyond providing a reliable assignment for the above mentioned characteristic Raman lines, the theoretical spectrum allowed us to carry out an overview of the whole observed data of Phe in aqueous solution. Copyright © 2013 John Wiley & Sons, Ltd.

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“…The Raman signature of the Δ-domain at 10 °C exhibits several features diagnostic of a highly α-helical secondary structure, including amide I (1649 cm −1 ), amide III (1272–1300 cm −1 ) and skeletal mode (939 cm −1 ) 18,26,27. Quantitative analysis of the secondary structure by decomposition of the amide I band28 indicates 90 ± 5% α-helix, 10 ± 1% β-strand and negligible irregular structure.…”

Section: Results and Interpretationmentioning

confidence: 99%

Unfolding Thermodynamics of the Δ-Domain in the Prohead I Subunit of Phage HK97: Determination by Factor Analysis of Raman Spectra

Němeček

Overman

Hendrix

et al. 2009

Journal of Molecular Biology

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SUMMARYAn early step in the morphogenesis of the double-stranded DNA bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (~70%), median melting temperature (T m = 58 °C), enthalpy (ΔH m = 50 ± 5 kcal·mol −1 ), entropy (ΔS m = 150 ± 10 cal·mol −1 ·K −1 ) and average cooperative melting unit (〈n c 〉 ~3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (~90% α-helix), exhibits diminished thermostability (T m = 40 °C, ΔH m = 27 ± 2 kcal·mol −1 , ΔS m = 86 ± 6 cal·mol −1 ·K −1 ) and noncooperative unfolding (〈n c 〉 ~ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.

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Raman Spectroscopy of Proteins

Cited by 47 publications

References 125 publications

Thermostable lipase fromGeobacillussp. Iso5: Bioseparation, characterization and native structural studies

Thermostable lipase fromGeobacillussp. Iso5: Bioseparation, characterization and native structural studies

Characteristic Raman lines of phenylalanine analyzed by a multiconformational approach

Unfolding Thermodynamics of the Δ-Domain in the Prohead I Subunit of Phage HK97: Determination by Factor Analysis of Raman Spectra

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