Overestimated accuracy of circular dichroism in determining protein secondary structure

Lin, Kailei; Yang, Huayan; Gao, Zhengya; Li, Feng; Yu, Shaoning

doi:10.1007/s00249-013-0896-y

Cited by 10 publications

(14 citation statements)

References 49 publications

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“…The spectra reflect the n-π* (222 nm) and low energy component of the π-π* (208.5 nm) transitions of the proteins, and the secondary structure, primarily α-helix, appears less in SL-CaM than in CaM. However, CD spectroscopy may not be a highly reliable method for determining secondary structures in proteins [54]. With SL-CaM, in particular, the nitroxide moiety is a chromophore in the far ultraviolet, and its contribution to CD is unknown.…”

Section: Resultsmentioning

confidence: 99%

Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

Bowman

Puett

2014

Protein J

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Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca2+)4-form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2-3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca2+ to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca2+ binding. The midpoint of the Ca2+-mediated transition determined by EPR occurred at a higher Ca2+ concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [Ca2+)4-CaM], contains a compact intermediate corresponding to [Ca2+)2-CaM], and the present results suggest that the spin probes are reporting on Ca2+ binding to the last two sites in the N-terminal domain, i.e. for the [Ca2+)2-CaM] → [Ca2+)4-CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca2+-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.

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Section: Resultsmentioning

confidence: 99%

Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

Bowman

Puett

2014

Protein J

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“…), the helical content predicted by CD is significantly lower than that exhibited by X-ray crystallography or Fourier transform infrared solution spectroscopy. 42 Inaccuracies in CD measurements can arise from the presence of scattering particles or protein concentrations that are too high or too low. Also, data collected in buffers at physiological ionic strength (0.150 M NaCl) can show a signal fluctuation around 190 nm due to Cl − ions that compromise the accuracy of the protein secondary structural prediction.…”

Section: Discussionmentioning

confidence: 99%

“…Also, data collected in buffers at physiological ionic strength (0.150 M NaCl) can show a signal fluctuation around 190 nm due to Cl − ions that compromise the accuracy of the protein secondary structural prediction. 42 Variability and overlap in the positions of the protein absorption bands as well as limited reference data reflect additional challenges for the empirical analysis of CD data. 42 It is likely that these factors contribute to the discrepancy in secondary structure content determined by CD spectroscopic and crystallographic methods.…”

Section: Discussionmentioning

confidence: 99%

X-ray Crystal Structure of Bovine 3 Glu-Osteocalcin

2013

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The 3 Glu form of osteocalcin (3 Glu-OCN) is increased in serum during low vitamin K intake or oral anticoagulant use (warfarin). Previous reports using circular dichroism show it is less structured than 3 Gla Ca2+-osteocalcin and does not bind strongly to bone mineral. Recent studies have suggested a role for 3 Glu-OCN as a potential regulator of glucose metabolism. A G-protein-coupled receptor, GPRC6a, found in the pancreas and testes was identified as the putative osteocalcin receptor. The purpose of this study is to determine the high-resolution structure of bovine 3 Glu-OCN, using X-ray crystallography, to understand molecular interactions with mineral and the GPRC6a receptor. Diffraction quality crystals of thermally decarboxylated bovine osteocalcin were grown, and the crystal structure was determined to 1.88 Å resolution. The final refined structure contained residues 17–47 and, like 3 Gla Ca2+-OCN, consisted of three α-helices surrounding a hydrophobic core, a C23–C29 disulfide bond between two of the helices, and no bound Ca2+. Thus, the helical structure of 3 Glu-OCN is Ca2+-independent but similar to that of 3 Gla Ca2+-OCN. A reduced level of mineral binding could result from a lower number of Ca2+ coordinating ligands on 3 Glu-OCN. The structure suggests the GPRC6a receptor may respond to helical osteocalcin and will aid in providing molecular mechanistic insight into the role of 3 Glu-OCN in glucose homeostasis.

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“…Curve-fitting of the Fourier transform infrared (FTIR) spectrum of a protein in the 1700–1600 cm −1 amide I region is widely used in the quantitative analysis of protein secondary structures [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. When implementing the FTIR curve-fitting approach, resolution-enhancement treatments with second derivative and Fourier self-deconvolution (FSD) are commonly used to resolve the overlapped component peaks corresponding to different secondary structures from the often featureless amide I band [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18].…”

Section: Introductionmentioning

confidence: 99%

“…When implementing the FTIR curve-fitting approach, resolution-enhancement treatments with second derivative and Fourier self-deconvolution (FSD) are commonly used to resolve the overlapped component peaks corresponding to different secondary structures from the often featureless amide I band [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. These two techniques offer two important fitting parameters, peak number and peak frequency.…”

Section: Introductionmentioning

confidence: 99%

A New Criterion to Evaluate Water Vapor Interference in Protein Secondary Structural Analysis by FTIR Spectroscopy

Zou

2014

IJMS

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Second derivative and Fourier self-deconvolution (FSD) are two commonly used techniques to resolve the overlapped component peaks from the often featureless amide I band in Fourier transform infrared (FTIR) curve-fitting approach for protein secondary structural analysis. Yet, the reliability of these two techniques is greatly affected by the omnipresent water vapor in the atmosphere. Several criteria are currently in use as quality controls to ensure the protein absorption spectrum is negligibly affected by water vapor interference. In this study, through a second derivative study of liquid water, we first argue that the previously established criteria cannot guarantee a reliable evaluation of water vapor interference due to a phenomenon that we refer to as sample’s absorbance-dependent water vapor interference. Then, through a comparative study of protein and liquid water, we show that a protein absorption spectrum can still be significantly affected by water vapor interference even though it satisfies the established criteria. At last, we propose to use the comparison between the second derivative spectra of protein and liquid water as a new criterion to better evaluate water vapor interference for more reliable second derivative and FSD treatments on the protein amide I band.

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Overestimated accuracy of circular dichroism in determining protein secondary structure

Cited by 10 publications

References 49 publications

Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

X-ray Crystal Structure of Bovine 3 Glu-Osteocalcin

A New Criterion to Evaluate Water Vapor Interference in Protein Secondary Structural Analysis by FTIR Spectroscopy

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