Circular Dichroism of Protein–Nucleic Acid Interactions

Gray, David B.

doi:10.1002/9781118120392.ch19

Cited by 6 publications

(8 citation statements)

References 50 publications

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“…We recorded spectra of isolated DNA sites, E2C-16, and the 1:1 protein−DNA complexes. The CD spectra of free and bound BS1 and BS2 show a positive peak at 275 nm, a crossover at 260 nm, and a negative peak at 245 nm (Figure S1 of the Supporting Information ), characteristic of B-form DNA ( 53 , 63 , 64 ), as expected from structural data ( 44 , 45 ). We calculated difference CD and absorbance spectra by subtracting the individual spectra of the free DNA and protein from the spectra of the corresponding complexes.…”

Section: Resultssupporting

confidence: 60%

“…Formation of E2C−DNA complexes induces bending of the DNA toward the minor groove and small rearrangements in the protein, as indicated by crystallographic evidence ( 44 , 45 ) and solution measurements ( 38 , 54 ). Because of the large difference in extinction coefficients in favor of DNA, the contribution of the protein to the spectra is minor and easily subtractable in absorbance and negligible in near-UV CD, which allows us to determine that spectral changes correspond mostly to DNA conformation ( 53 , 63 ). Thus, we may attribute the changes in CD and absorbance spectra to DNA bending and torsion.…”

Section: Resultsmentioning

confidence: 99%

“…We recorded spectra for the isolated DBS and for the ternary E2C−DBS−E2C complex. The CD spectra of the free DBS and of the ternary complex exhibit a positive peak at 275 nm, a crossover at 260, and a negative peak at 245 nm (Figure S5 of the Supporting Information ), characteristic of B-form DNA ( 53 , 63 , 64 ). This shows that the DNA of DBS forms a B type of helix both in its free form and in the ternary complex with E2C-16.…”

Section: Resultsmentioning

confidence: 99%

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Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Dellarole

Sánchez

2010

Biochemistry

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Binding cooperativity guides the formation of protein−nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic−entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

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Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Dellarole

Sánchez

2010

Biochemistry

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“…Theoretical calculation shows that ECD signal of polynucleotides mainly origins from the exciton coupling interaction between bases stacked in asymmetric structure of higher ordering like helices, than from the base-sugar interaction in monomeric nucleotides, as well as it arises for the most part from the ππ* transitions, rather than nπ* ones [ 54 ]. Thus, perturbation of a nucleic acid ECD profile, that appears due to binding of a protein, generally reflects changes in short-range base–base interactions, providing confirmation for transitions between secondary structures (A↔B, B↔Z) or alternations in base pairing, base stacking, or bending of a DNA or RNA duplex [ 55 ].…”

Section: Discussionmentioning

confidence: 99%

Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

Halat

Klimek-Chodacka

Orleanska

et al. 2021

IJMS

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The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

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“…VUVCD spectroscopy as well as conventional CD can also be applied to various systems such as DNA–protein interactions [ 152 – 154 ] and DNA damage [ 155 – 157 ]. Since these approaches provide important structural information that is closely related to the genetic and physiological functions of nucleic acids, VUVCD spectroscopy will certainly also be useful in genomic structural biology.…”

Section: Structural Analysis Of Nucleic Acidsmentioning

confidence: 99%

Synchrotron-radiation vacuum-ultraviolet circular dichroism spectroscopy in structural biology: an overview

Gekko

2019

BIOPHYSICS

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Circular dichroism spectroscopy is widely used for analyzing the structures of chiral molecules, including biomolecules. Vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy using synchrotron radiation can extend the short-wavelength limit into the vacuum-ultraviolet region (down to ~160 nm) to provide detailed and new information about the structures of biomolecules in combination with theoretical analysis and bioinformatics. The VUVCD spectra of saccharides can detect the high-energy transitions of chromophores such as hydroxy and acetal groups, disclosing the contributions of inter- or intramolecular hydrogen bonds to the equilibrium configuration of monosaccharides in aqueous solution. The roles of hydration in the fluctuation of the dihedral angles of carboxyl and amino groups of amino acids can be clarified by comparing the observed VUVCD spectra with those calculated theoretically. The VUVCD spectra of proteins markedly improves the accuracy of predicting the contents and number of segments of the secondary structures, and their amino acid sequences when combined with bioinformatics, for not only native but also nonnative and membrane-bound proteins. The VUVCD spectra of nucleic acids confirm the contributions of the base composition and sequence to the conformation in comparative analyses of synthetic poly-nucleotides composed of selected bases. This review surveys these recent applications of synchrotron-radiation VUVCD spectroscopy in structural biology, covering saccharides, amino acids, proteins, and nucleic acids.

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Circular Dichroism of Protein–Nucleic Acid Interactions

Cited by 6 publications

References 50 publications

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

Synchrotron-radiation vacuum-ultraviolet circular dichroism spectroscopy in structural biology: an overview

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