Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication

Compton, Larry A.; Johnson, W. Curtis

doi:10.1016/0003-2697(86)90241-1

Cited by 537 publications

(410 citation statements)

References 13 publications

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“…Prior to spectral acquisition, the spectropolarimeter was fluxed with dry N 2 for at least 30 min. ∆ values (M -1 ‚cm -1 ) were calculated from the observed instrument output (φ, in millidegrees), the protein concentration (c, in molar), and the number of peptide bonds (n), according to the formula: ∆ ) φ/3300cn (28).…”

Section: Methodsmentioning

confidence: 99%

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

et al. 2001

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Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

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Section: Methodsmentioning

confidence: 99%

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

et al. 2001

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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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“…This spectrum is similar to protonated polylysine in the random coil state (Brahms et al, 1977) and suggests that RED in solution exists primarily in a disordered conformation with little secondary structure. A &turn structure is the only other derived structure (Compton & Johnson, 1986) that resembles that of the RED. This spectrum and structural interpretation are markedly different than for that observed for the epidermal growth factor (EGF) receptor extracellular domain, which has significant amounts of regular secondary structure (Greenfield et al, 1989).…”

Section: CDmentioning

confidence: 99%

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor

Timm

Vissavajjhala²,

Ross³

et al. 1992

Protein Science

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Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and C D change. During heating at 100 "C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

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Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication

Cited by 537 publications

References 13 publications

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

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

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor

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