Using circular dichroism spectra to estimate protein secondary structure

Greenfield, Norma J.

doi:10.1038/nprot.2006.202

Cited by 3,234 publications

(2,722 citation statements)

References 63 publications

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“…Both PaHisGS and PaHisZ yielded CD spectra typical of folded proteins ( Figure 6E). 49 Thermal denaturation data were fitted to eq 6 (Table S1). Hence there is no loss in affinity between the two proteins at 308 K to account for the decrease in PaATPPRT kcat.…”

Section: Thermal Denaturation Of Pahisgs and Pahisz To Interrogate Tmentioning

confidence: 99%

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Stroek

Talbot²,

Glok³

et al. 2017

Biochemistry

112

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ATP-phosphoribosyltransferase (ATPPRT) catalyses the first step in histidine biosynthesis, the condensation of ATP and 5-phospho--D-ribosyl-1-pyrophosphate to generate N 1 -(5-phospho--D-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain, and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was solved at 2.34-Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-to 4-fold increase in kcat.Reaction profiles for both enzymes are indistinguishable by 31 P-NMR, indicating that the same reaction is catalysed. Temperature dependence of kcat shows deviation from Arrhenius behaviour at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 K and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterisation of a psychrophilic ATPPRT. 4Adenosine 5ʹ-triphosphate phosphoribosyltransferase (ATPPRT) (EC 2.4.2.17) catalyses the reversible Mg 2+ -dependent reaction between adenosine 5ʹ-triphosphate (ATP) and(PR-ATP) and inorganic pyrophosphate (PPi) (Scheme 1), the first step in histidine biosynthesis. 1 The chemical equilibrium of the reaction strongly favours reactants, 2 and the enzyme is allosterically inhibited by histidine. 1 In addition to being a model for understanding allostery, 2-4 ATPPRT is of biotechnological interest as a tool for histidine production, provided that histidine feedback inhibition can be overcome. [5][6][7] Two forms of ATPPRT, encoded by the hisG gene, are found in nature. Fungi, plants, and most bacteria possess a long, homo-hexameric protein, HisGL, each subunit consisting of two domains that make up the catalytic core and a C-terminal regulatory domain that mediates feedback inhibition by histidine. 8 Some bacteria and archaea have a short version of the protein, HisGS, which lacks the C-terminal regulatory domain and is insensitive to histidine. In these organisms, a catalytically inactive regulatory protein, HisZ, the product of the hisZ gene, is present. 9 HisZ, which shares a common ancestry with histidyl-tRNA synthetase (HisRS), binds HisGS to form ...

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Section: Thermal Denaturation Of Pahisgs and Pahisz To Interrogate Tmentioning

confidence: 99%

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Stroek

Talbot²,

Glok³

et al. 2017

Biochemistry

112

View full text Add to dashboard Cite

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“…Peptide concentrations were determined by UV absorption at 280 nm (ε(Trp) = 5690 mol -1 cm -1 ; ε(Tyr) = 1280 mol -1 cm -1 ). 45 Peptide solutions were prepared in PBS, and examined in 5 mm quartz cuvettes. Thermal denaturation experiments were performed by ramping temperature from 5°C to 90°C at a rate of 10°C/h.…”

Section: Sedimentation-equilibrium Experiments By Analytical Ultracenmentioning

confidence: 99%

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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The α-helical coiled coil is one of the best-studied protein–protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix–helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

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“…However, in the presence of SUVs formed from lipid extracts of S. aureus membranes, at pH 6, pH 7 and pH 8, CD spectra showed minima at 221 -222 and 209 -210 nm ( Figure 3), which is indicative of α-helical structure in MH5 55 . Analysis of these CD spectra revealed that the levels of α-helicity were around 48 % at pH 8, but increased to 58 % as pH was decreased to pH 6, showing clearly that MH5 is able to form an α-helical structure in the presence of an asymmetric interface that is enhanced by low pH (Table 1).…”

Section: Secondary Structure Of Mh5 In the Presence Of Lipid Mimics Omentioning

confidence: 99%

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

et al. 2016

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Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m–1) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m–1 and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism’s growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 μM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45–50%) and showed similarities to the “Carpet” mechanism based on its ability to increase the rigidity (C s –1 = 109.94 mN m–1) and thermodynamic stability (ΔG mix = −3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretical analysis, this mechanism might involve the use of a tilted peptide structure, and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R 2 ∼ 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG-induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of hemolytic activity (<2% hemolysis) and merits further investigation as a potential template for development as an antistaphylococcal agent in medically and biotechnically relevant areas.

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Using circular dichroism spectra to estimate protein secondary structure

Cited by 3,234 publications

References 63 publications

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

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