Estimation of protein secondary structure and error analysis from circular dichroism spectra

Stokkum, Ivo H. M. van; Spoelder, H.J.W.; Bloemendal, Michael; Grondelle, Rienk van; Groen, Frans C. A.

doi:10.1016/0003-2697(90)90396-q

Cited by 478 publications

(372 citation statements)

References 12 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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“…Secondary structure analyses were carried out using the DichroWeb analysis server [19]. Values from the algorithms CONTINLL [20,21], SELCON [22] and CDDSTR [22] (using reference set SMP180 [23]) were averaged and their standard deviations reported. SMP180 is a new reference data set specifically devised for use with membrane proteins.…”

Section: Srcd Spectroscopymentioning

confidence: 99%

“…3C and F). a Values from the algorithms CONTINLL [20,21], SELCON [22] and CDDSTR [22] (using reference set SMP180 [23]). The values reported are the average of the three algorithms, with ± reflecting the standard deviation between the values calculated by the different methods.…”

Section: Spectral Analyses Of Thermaldenaturation Of Solubilised Nakmentioning

confidence: 99%

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

Miles

Fedosova

Hoffmann

et al. 2013

Biochemical and Biophysical Research Communications

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a b s t r a c tCardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

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Estimation of protein secondary structure and error analysis from circular dichroism spectra

Cited by 478 publications

References 12 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

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

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