Biomedical subjects

Jannette Carey

Publications and source records attributed to Jannette Carey.

9 recordsLinked to original sources

PubMed2007

Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA.

WrbA is an oligomeric flavodoxin-like protein that binds one molecule of flavin mononucleotide (FMN) per monomer and whose redox activity is implicated in oxidative stress defense. WrbA thermostability and oligomerization in the presence and absence of bound FMN were investigated using complementary biophysical methods. Infrared spectroscopy indicates similar structures for apo and holoWrbA. FMN binding has a dramatic effect on WrbA thermal stability, shifting the Tm by approximately 40 degrees C. Upon denaturation, the protein forms insoluble aggregates that lack native secondary structure and have no bound FMN. Circular dichroism (CD) reveals that the thermal unfolding of apo and holoWrbA proceeds via the formation of an aggregation-prone intermediate that retains substantial secondary structure but has lost the native configuration of the active site. This intermediate persists in solution up to 100 degrees C at micromolar concentrations. A similar partially folded state is populated during chemical denaturation with guanidinium chloride, but accumulation of the intermediate is evident only in the absence of FMN. The results also suggest that WrbA maintains some interaction with FMN in its partially folded state, despite the loss of the induced CD signal of FMN. On the basis of these data, the unfolding process can be depicted as follows: native holoprotein --> holointermediate --> apointermediate --> insoluble aggregate. Mass spectrometry shows that FMN promotes WrbA association into tetramers, which are more thermoresistant than dimers or monomers, suggesting that multimerization underlies the FMN effect on WrbA thermostability. This study illustrates the utility of analyzing conformational transitions and intermolecular interactions using methods that probe the liquid, solid, and gas phases.

Circular Dichroism↗

PubMed2006

Spectroelectrochemical investigation of a flavoprotein with a flavin-modified gold electrode.

A flavin-modified gold electrode was developed in order to catalyze the electrochemical oxidoreduction of flavoproteins. Surface modification was carried out by a two-step procedure. In the first step a mixed self-assembled monolayer obtained by adsorption of activated and nonactivated 3,3'-dithiopropionic acid (free acid and N-succinimidyl ester) was formed, followed by the covalent attachment of a N(10)-hexylamino-alkylated flavin derivative via an amide bond in the second step. The electrochemical properties of the flavin-modified electrode are presented and discussed. The redox potential of the attached flavin was measured at various pH values and the electron-transfer rate constant between electrode and flavin was determined as k0 = 5 s(-1) independent of pH. The flavin-modified electrode was successfully applied to the electrochemical and spectroelectrochemical investigation of the flavoprotein WrbA from Escherichia coli that shows some structural similarities to flavodoxins. It is concluded that the electron transfer "electrode --> flavin --> flavoprotein" occurs by a two-step hopping mechanism where the first step is rate determining. Kinetic details are discussed. Furthermore, it turned out that, in contrast to flavodoxins, where the semiquinone state is stabilized, WrbA rapidly takes up two electrons, directly leading to the fully reduced form. The presented electrode surface modification may generally lend itself for spectroelectrochemical investigations of flavoproteins.

Adsorption↗

PubMed2005

Assembly of the hexameric Escherichia coli arginine repressor investigated by nano-electrospray ionization time-of-flight mass spectrometry.

The arginine repressor (ArgR) from Escherichia coli regulates genes for L-arginine metabolism and is a required recombination factor for colE1 plasmid replication. Both functions require binding of L-arginine to the protein. In this work, nano-electrospray ionization time-of-flight mass spectrometry (nano-ESI-TOFMS) is used to study conformational and oligomeric states of intact ArgR and its isolated structural domains. In agreement with X-ray diffraction studies, it is shown that ArgR oligomerizes to form hexamers in both the presence and absence of L-arginine, and the basic unit of oligomerization appears to be the trimer. Higher-order assembly into dodecamers is also detected. The isolated C-terminal domain is found to associate into trimers and hexamers whereas the N-terminal domain is detected in its monomeric form. The observed species distributions suggest a role for the N-terminal domain in hexamer stabilization.

Escherichia coli↗

PubMed2005

Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel.

PURPOSE: Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology. METHODS: The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology. RESULTS: All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication. CONCLUSIONS: The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

Amino Acid Sequence↗

PubMed2004

Asymmetric allosteric activation of the symmetric ArgR hexamer.

Hexameric arginine repressor, ArgR, bound to L-arginine serves both as the master transcriptional repressor/activator at diverse regulons in a wide range of bacteria and as a required cofactor for resolution of ColE1 plasmid multimers. Multifunctional ArgR is thus unusual in possessing features of specific gene regulators, global regulators, and non-specific gene organizers; its closest functional analog is probably CAP, the cyclic AMP receptor/activator protein. Isothermal titration calorimetry, surface plasmon resonance, and proteolysis indicate that binding of a single L-argine [corrected] per ArgR hexamer triggers a global conformation [corrected] change and resets the affinities of the remaining five sites, making them 100-fold weaker. The analysis suggests a novel thermodynamic signature for this mechanism of activation.

Allosteric Regulation↗

PubMed2004

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin.

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.

Calorimetry↗

PubMed2004

Biophysical properties of regions flanking the bHLH-Zip motif in the p22 Max protein.

The Max protein is the central dimerization partner in the Myc-Max-Mad network of transcriptional regulators, and a founding structural member of the family of basic-helix-loop-helix (bHLH)-leucine zipper (Zip) proteins. Biologically important regions flanking its bHLH-Zip motif have been disordered or absent in crystal structures. The present study shows that these regions are resistant to proteolysis in both the presence and absence of DNA, and that Max dimers containing both flanking regions have significantly higher helix content as measured by circular dichroism than that predicted from the crystal structures. Nuclear magnetic resonance measurements in the absence of DNA also support the inferred structural order. Deletion of both flanking regions is required to achieve maximal DNA affinity as measured by EMSA. Thus, the previously observed functionalities of these Max regions in DNA binding, phosphorylation, and apoptosis are suggested to be linked to structural properties.

3' Flanking Region↗

PubMed2004

E. coli trp repressor forms a domain-swapped array in aqueous alcohol.

The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 A X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.

2-Propanol↗

PubMed2002

Three-dimensional solution NMR structure of Apo-L75F-TrpR, a temperature-sensitive mutant of the tryptophan repressor protein.

L75F-TrpR is a temperature-sensitive mutant of the tryptophan repressor protein of Escherichia coli in which surface-exposed residue leucine 75 in the DNA binding domain is replaced with phenylalanine. Biochemical and biophysical studies had suggested global alterations in dynamics for L75F-TrpR, although the structure was apparently similar to that of wild-type TrpR. Herein, we report the three-dimensional solution structure of apo-L75F-TrpR determined by multidimensional ((1)H, (15)N, and (13)C) solution NMR spectroscopy. An ensemble of structures was generated from 769 unique NOE-based distance restraints, 68 dihedral angle restraints, and 62 hydrogen bond distance restraints. Apo-L75F-TrpR exhibits a three-dimensional (3D) fold very similar to that of apo-WT-TrpR, with a dimeric core of four alpha-helices (A-C and F) from each subunit, and less well-defined D and E helical regions of the DNA binding domains. Despite their many similarities, wild-type and mutant proteins display significant chemical shift differences, one cluster of which is in the B-C turn, too distant to be ascribed solely to ring current effects from Phe75. Differences in NOE patterns and amide proton exchange rates are also observed in the B-C turn region. The data provide evidence that this point mutation exerts local effects on structure and stability in the DNA binding domain, and propagates long-range effects through the tertiary structure.

Amino Acid Sequence↗