Macromolecular Interactions: Light Scattering

Folta‐Stogniew, Ewa

doi:10.1002/9780470015902.a0003143

Cited by 21 publications

(28 citation statements)

References 38 publications

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“…These values were much higher than that obtained during mass spectrometry for the monomer (43 kDa, see below), thus suggesting an abnormal SEC migration of this form, probably depending on the presence of the PG domain with a non-globular structure. This hypothesis was confirmed by LS analysis of the eluted SEC fractions, which was performed under native conditions (30). In this case, P1 and P2 showed an average molecular mass value of 86 and 44 kDa, respectively (shown with dots in Fig.…”

Section: Resultssupporting

confidence: 50%

Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Hilvo¹,

Baranauskienė²,

Salzano

et al. 2008

Journal of Biological Chemistry

275

305

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Section: Resultssupporting

confidence: 50%

Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Hilvo¹,

Baranauskienė²,

Salzano

et al. 2008

Journal of Biological Chemistry

275

305

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“…Knowing the ratio of detergent bound per protein, the mass of the protein without micelle is calculated according to Equation 1, (20,21), where M B is the buoyant mass, Ј is the buoyant term that is composed of the different components of the analyzed complex, M P is the molecular mass of the protein, P is the partial specific volume of the protein, Det is the partial specific volume of the detergent (22), ␦ is the ratio of detergent bound per protein in g g Ϫ1 , and is the density of the buffer. Static Light Scattering-For size determination by static light scattering, size exclusion chromatography was coupled with continuous laser light scattering, refractive index, and ultraviolet detection of the eluent as described (23). Na ϩ -NQR was separated on a Superdex 200 10/300 column (GE Healthcare) connected to an Agilent 1100 HPLC system at 20°C, and protein was eluted with a flow rate of 0.5 ml min Ϫ1 using gel filtration buffer.…”

Section: Methodsmentioning

confidence: 99%

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Casutt

Huber

Brunisholz³

et al. 2010

Journal of Biological Chemistry

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The sodium ion-translocating NADH:quinone oxidoreductase (Na ؉ -NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na ؉ across the bacterial membrane. The Na ؉ -NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na ؉ -NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na ؉ -NQR is discussed.Pathogenic strains of the water-borne bacterium Vibrio cholerae cause the diarrheal disease cholera. By the help of a respiratory Na ϩ pump, V. cholerae maintains an electrochemical Na ϩ gradient across the inner membrane to drive central processes like flagellar rotation, nutrient uptake, and detoxification. The respiratory Na ϩ pump, also called Na2 is a membrane-bound enzyme complex composed of six subunits (NqrABCDEF) (1, 2). NqrA is a peripheral subunit, NqrC and NqrF are predominantly hydrophilic yet anchored to the membrane, whereas the hydrophobic NqrB, NqrD, and NqrE subunits each comprise several transmembrane helices. The Na ϩ -NQR contains at least five distinct redox centers participating in electron transfer from NADH to substrate quinone: noncovalently bound FAD, a 2Fe-2S cluster, covalently bound FMNs, and noncovalently bound riboflavin (3-5). In addition, ubiquinone-8 (Q 8 ) was co-purified with the Na ϩ -NQR (6). Binding sites for FAD, 2Fe-2S cluster, and substrate NADH are located on NqrF, which does not directly participate in Na ϩ -transport but represents the electron input module of the complex catalyzing the NADH dehydrogenase reaction (7,8). The covalently bound FMN residues are attached via phosphodiester bonds to Thr-236 in subunit NqrB and Thr-225 in subunit NqrC, respectively (9, 10). The subunit(s) responsible for riboflavin and quinone binding have not been identified yet. Riboflavin was detected in a subcomplex composed of NqrA, -B, -C, and -F but was not present in the isolated A and F subunits (3). Furthermore, there is evidence that NqrB participates in the quinone reduction step (11).By analyzing the flavin cofactors in defined subcomplexes of the Na ϩ -NQR from V. cholerae, we show that riboflavin is localized on subunit NqrB. Analysis of the molar size and subunit composition of the holo-complex reveals that the Na ϩ -NQR contains one copy of each subunit and a total ...

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“…Data analysis to determine molecular masses was performed with ASTRA software (51). Experiments were conducted at the Keck Foundation Biotechnology Resource Laboratory at Yale University (52).…”

Section: Site-directed Mutagenesis and Cloning-primersmentioning

confidence: 99%

Intensity of Deoxycytidine Deamination of HIV-1 Proviral DNA by the Retroviral Restriction Factor APOBEC3G Is Mediated by the Noncatalytic Domain

Feng¹,

Chelico

2011

Journal of Biological Chemistry

154

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APOBEC3G is a single-stranded (ss) DNA deaminase that restricts replication of HIV-1 by inducing viral genome mutagenesis through deamination of cytosine to uracil on HIV-1 cDNA. APOBEC3G has polydisperse oligomeric states and deaminates ssDNA processively through jumping and sliding. APOBEC3G has a catalytically inactive N-terminal CD1 domain that mediates processivity and an active C-terminal CD2 domain that catalyzes deaminations. Here, we assess the determinants of APOBEC3G deamination efficiency mediated by the CD1 domain by comparing native APOBEC3G and two CD1 mutants, a monomeric mutant (F126A/W127A) and a clinical mutant associated with high viral loads (H186R). Biochemical assays on ssDNA or partially dsDNA and with a reconstituted HIV replication system demonstrate that both mutants of Apo3G have altered DNA scanning properties in either jumping (F126A/W127A) or sliding (H186R), which results in decreased abilities to induce mutagenesis during reverse transcription. The data reveal a functionality for Apo3G oligomers in deamination and provide the first biochemical characterization of the clinical mutant H186R. The data demonstrate that the balance between the jumping and sliding of Apo3G is needed for efficient mutational inactivation of HIV-1.

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Macromolecular Interactions: Light Scattering

Cited by 21 publications

References 38 publications

Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Intensity of Deoxycytidine Deamination of HIV-1 Proviral DNA by the Retroviral Restriction Factor APOBEC3G Is Mediated by the Noncatalytic Domain

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