Biomedical subjects

Timothy M Logan

Publications and source records attributed to Timothy M Logan.

15 recordsLinked to original sources

PubMed2006

An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells.

It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with 15N and carbohydrates with 13C by NMR spectroscopy.

Amino Acids↗

PubMed2006

Metal-linked dimerization in the iron-dependent regulator from Mycobacterium tuberculosis.

The iron-dependent regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. The natural ligand for IdeR is Fe(II), but Ni(II), Co(II), Cd(II), Mn(II), and Zn(II) also bind to and activate the protein in vitro. Protein activation by metal is a complex process involving metal-induced folding of the N-terminal domain, changes in the interaction between the N- and C-terminal domains, and the formation of homodimers. Here, we investigate the energetics of dimerization and metal binding in IdeR. The dimerization energetics were determined as a function of metal binding using equilibrium analytical ultracentrifugation. The equilibrium dimer dissociation constant of apo-IdeR was 4.0 microM at 20 degrees C. The dissociation constant decreased to 0.5 microM in the presence of one equivalent of Ni(II)Cl(2) and decreased further (K(d) << 50 nM) in the presence of excess Ni(II). IdeR contains two tryptophan residues. The addition of Ni(II) induced changes in fluorescence intensity and emission maximum of the tryptophan residues that strongly depended on protein concentration. At low IdeR concentration, fluorescence was enhanced at low metal-to-protein ratios but was quenched at high metal-to-protein ratios. At high IdeR concentration, metal binding resulted only in fluorescence quenching. The fluorescence enhancement at low protein concentrations was buffer-dependent and required the presence of both tryptophans. Metal binding affinity was measured quantitatively using equilibrium dialysis. The results showed strongly positive cooperative binding of three equivalents of metal per monomer with an average apparent dissociation constant of 2.2 +/- 0.3 microM and a Hill coefficient of 2. Metal binding was not cooperative in an IdeR variant that showed reduced affinity for dimer formation. The results of this study establish the positive cooperative nature of metal binding by IdeR and suggest that dimerization is a major contributor to cooperative binding. The strong coupling between metal binding and dimerization places specific constraints on the activation mechanism.

Amino Acid Substitution↗

PubMed2006

Mn(II) binding by the anthracis repressor from Bacillus anthracis.

The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 microM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s(-1). Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.

Bacillus anthracis↗

PubMed2005

Effects of pH, salt, and macromolecular crowding on the stability of FK506-binding protein: an integrated experimental and theoretical study.

Environmental variables can exert significant influences on the folding stability of a protein, and elucidating these influences provides insight on the determinants of protein stability. Here, experimental data on the stability of FKBP12 are reported for the effects of three environmental variables: pH, salt, and macromolecular crowding. In the pH range of 5-9, contribution to the pH dependence of the unfolding free energy from residual charge-charge interactions in the unfolded state was found to be negligible. The negligible contribution was attributed to the lack of sequentially nearest neighboring charged residues around groups that titrate in the pH range. KCl lowered the stability of FKBP12 and the E31Q/D32N double mutant at small salt concentrations but raised stability after approximately 0.5 M salt. Such a turnover behavior was accounted for by the balance of two opposing types of protein-salt interactions: the Debye-Hückel type, modeling the response of the ions to protein charges, favors the unfolded state while the Kirkwood type, accounting for the disadvantage of the ions moving toward the low-dielectric protein cavity from the bulk solvent, disfavors the unfolded state. Ficoll 70 as a crowding agent was found to have a modest effect on protein stability, in qualitative agreement with a simple model suggesting that the folded and unfolded states are nearly equally adversely affected by macromolecular crowding. For any environmental variable, it is the balance of its effects on the folded and unfolded states that determines the outcome on the folding stability.

Ficoll↗

PubMed2005

Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Amino Acid Substitution↗

PubMed2005

Prolylpeptide binding by the prokaryotic SH3-like domain of the diphtheria toxin repressor: a regulatory switch.

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.

Amino Acid Sequence↗

PubMed2005

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Amino Acid Sequence↗

PubMed2004

Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide.

In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide.

Amino Acid Sequence↗

PubMed2004

IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.

Amino Acid Sequence↗

PubMed2004

Genetic and biophysical studies of diphtheria toxin repressor (DtxR) and the hyperactive mutant DtxR(E175K) support a multistep model of activation.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding. We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites. We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N- and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor. Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process. Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions.

Alanine↗

PubMed2004

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells.

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.

Amino Acid Sequence↗

PubMed2003

Identification of a key structural element for protein folding within beta-hairpin turns.

Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

Alanine↗

PubMed2003

Design of small volume HX and triple-resonance probes for improved limits of detection in protein NMR experiments.

Three- and four-frequency nuclear magnetic-resonance probes have been designed for the study of small amounts of protein. Both "HX" (1H, X, and 2H channels) and "triple-resonance" (1H, 15N, 13C, and 2H) probes were implemented using a single transmit/receive coil and multiple-frequency impedance matching circuits. The coil used was a six-turn solenoid with an observe volume of 15 microl. A variable pitch design was used to improve the B1 homogeneity of the coil. Two-dimensional HSQC spectra of approximately 1mM single labeled 15N- and double labeled 15N/13C-proteins were acquired in experimental times of approximately 2h. Triple-resonance capability of the small-volume triple-resonance probe was demonstrated by acquiring three-dimensional HNCO spectra from the same protein samples. In addition to enabling very small quantities of protein to be used, the extremely short pulse widths (1H = 4, 15N = 4, and 13C = 2 micros) of this particular design result in low power decoupling and wide-bandwidth coverage, an important factor for the ever-higher operating frequencies used for protein NMR studies.

Carbon Isotopes↗

PubMed2003

Accommodation of a highly symmetric core within a symmetric protein superfold.

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.

Amino Acid Sequence↗

PubMed2002

Glutamine 53 is a gatekeeper residue in the FK506 binding protein.

The effect of non-random conformational averaging in the urea-unfolded state on the folding pathway has been investigated in a variant of the FK506 binding protein with three additional residues at the amino terminus (FKBP(*)). Three mutations (asparagine, aspartate, and threonine) were introduced into position Q53 to enhance formation of non-native helix observed in this part of the protein in the urea-unfolded state. NMR analysis showed minor structural changes in the native state of each mutant, but additional medium-range alphaN(i,i+2) of each mutant nuclear Overhauser enhancements were observed in the urea-unfolded state that were not in FKBP(*), indicating that the mutations had a more substantial effect on the unfolded state ensemble than on the native state ensemble. Isothermal equilibrium denaturation measurements showed that the Q53T and Q53D mutants were destabilized, whereas the Q53N mutant was stabilized relative to FKBP(*) with little change in the equilibrium m values. The unfolding rates of Q53N and Q53T were similar to that of FKBP(*), but Q53D unfolded twice as fast as FKBP(*). In contrast, the mutations had a more pronounced effect on the refolding kinetics. Q53N refolded slightly faster and exhibited a kinetic folding intermediate similar to that of FKBP(*). The Q53D and Q53T mutants also refolded faster than FKBP(*) but lacked the folding intermediate, indicating that these mutants experienced a different folding trajectory and transition state than FKBP(*) and Q53N. The refolding kinetic Phi values were 0.74, 1.4 and 7.9 for Q53N, Q53T, and Q53D, respectively. The data point to Q53 functioning as a gatekeeper residue in the folding of FKBP(*). This study shows that perturbing the unfolded state ensemble via mutagenesis can provide insights into residues that play important roles in the folding pathway, and represents an attractive strategy for mapping the high-energy portions of the folding energy landscape.

Glutamine↗