Estimation of globular protein secondary structure from circular dichroism

Provencher, Stephen W.; Glockner, James F.

doi:10.1021/bi00504a006

Cited by 2,087 publications

(1,522 citation statements)

References 18 publications

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“…CD and difference spectra were fit by nonconstrained MLR 23 using a polypeptide reference set. 24 Spectra of the individual sRz and sRz1 proteins were also fit using the CONTINLL 25,26 and CDSSTR 27 programs available on the DICHROWEB server. 28 To determine the molar ratio of sRz to sRz1 in the complex, a 10 lM solution of sRz in 10 mM sodium phosphate, 100 mM NaCl, pH 7.5, at 25 C was titrated, in 2.5 lM increments, with a stock solution of sRz1 supplemented with 10 lM sRz.…”

Section: Circular Dichroism Measurementsmentioning

confidence: 99%

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

et al. 2010

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Phage holins and endolysins have long been known to play key roles in lysis of the host cell, disrupting the cytoplasmic membrane and peptidoglycan (PG) layer, respectively. For phages of Gram-negative hosts, a third class of proteins, the spanins, are involved in disrupting the outer membrane (OM). Rz and Rz1, the components of the lambda spanin, are, respectively, a class II inner membrane protein and an OM lipoprotein, are thought to span the entire periplasm by virtue of C-terminal interactions of their soluble domains. Here, the periplasmic domains of Rz and Rz1 have been purified and shown to form dimeric and monomeric species, respectively, in solution. Circular dichroism analysis indicates that Rz has significant alpha-helical character, but much less than predicted, whereas Rz1, which is 25% proline, is unstructured. Mixture of the two proteins leads to complex formation and an increase in secondary structure, especially alpha-helical content. Moreover, transmission electron-microscopy reveals that Rz-Rz1 complexes form large rod-shaped structures which, although heterogeneous, exhibit periodicities that may reflect coiledcoil bundling as well as a long dimension that matches the width of the periplasm. A model is proposed suggesting that the formation of such bundles depends on the removal of the PG and underlies the Rz-Rz1 dependent disruption of the OM.

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Section: Circular Dichroism Measurementsmentioning

confidence: 99%

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

et al. 2010

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“…For secondary structure predictions, spectra in the far-UV region (190-240 nm) were recorded at 22 C with 0.5 nm increments in a 0.5 mm quartz cell, and six scans were averaged for each spectrum. Secondary structure estimates of wild type and mutant MexR was made using DICHROWEB 33,51 and the algorithms CDSSTR, 52,53 CONTIN, 54,55 and Selcon3. 56 Thermal denaturation spectra were recorded at 210-240 nm with 5 C intervals from 15-85 C using a 0.5 mm quartz cell, 15 lM protein in 10 mM sodium phosphate buffer, pH 7.2.…”

Section: Circular Dichroism Experimentsmentioning

confidence: 99%

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

et al. 2010

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The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.

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“…The spectrum reveals molar ellipticity minima at 208 nm and 222 nm, indicative of the presence of α-helix secondary structure. Deconvolution of the spectrum with the Contin program of Provencher and Glöckner (11) indicated that, at pH 3, apoA-V is comprised of 32 % a-helix, 33 % ß-sheet, 16 ß-turn and 18 % random coil. Thus, unlike most members of the exchangeable apolipoprotein family, apoA-V displays significant amounts of ß structure.…”

Section: Apoa-v Interaction With Phospholipid Bilayer Vesicles Of DImentioning

confidence: 99%

Structure−Function Studies of Human Apolipoprotein A-V: A Regulator of Plasma Lipid Homeostasis

et al. 2003

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To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% α-helix, 33% β-sheet, 16% β-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 °C. Upon being cooled to ambient temperature from 85 °C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15−20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 ± 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.

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Estimation of globular protein secondary structure from circular dichroism

Cited by 2,087 publications

References 18 publications

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

Structure−Function Studies of Human Apolipoprotein A-V: A Regulator of Plasma Lipid Homeostasis

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