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Bryan A Krantz

Publications and source records attributed to Bryan A Krantz.

15 recordsLinked to original sources
PubMed2007

Characterizing protein folding transition States using Psi-analysis.

We discuss the implementation of Psi-analysis for the structural characterization of protein folding transition states. In Psi-analysis, engineered bi-histidine metal ion binding sites are introduced at surface positions to stabilize secondary and tertiary structures. The addition of metal ions stabilizes the interaction between the two known histidines in a continuous fashion. Measuring the ratio of transition state stabilization to that of the native state provides information about the presence of the metal binding site in the transition state. Psi-Analysis uses noninvasive surface mutations and does not require specialized equipment, so it can be readily applied to characterize the folding of many proteins. As a result, this method can provide a wealth of high-resolution quantitative data for comparison with theoretical folding simulations. Additionally, investigations of other biological processes also may utilize metal binding sites and Psi-analysis to detect conformational events during catalysis, assembly, and function.

Binding Sites↗
PubMed2006

Assembly and disassembly kinetics of anthrax toxin complexes.

Proteolytic activation of the protective antigen (PA) component of anthrax toxin allows it to self-associate into a ring-shaped homoheptamer, [PA(63)](7), which can bind the enzymatic components lethal factor (LF) and edema factor (EF). [PA(63)](7) is a pore-precursor (prepore), and under the low-pH conditions of the endosome, it forms a transmembrane pore that allows LF and EF to enter the cytosol. PA was labeled with donor and acceptor fluorescent dyes, and Förster resonance energy transfer was used to measure the assembly and disassembly kinetics of the prepore complex in solution. The dissociation rate constant for [PA(63)](7) was 1 x 10(-)(6) s(-)(1) (t(1/2) approximately 7 days). In contrast, a ternary complex containing the PA-binding domain of LF (LF(N)) bound to a PA(63) dimer composed of two nonoligomerizing mutants dissociated rapidly (t(1/2) approximately 1 min). Thus, the substantial decrease in the rate of disassembly of [PA(63)](7) relative to the ternary complex is due to the cooperative interactions among neighboring subunits in the heptameric ring. Low concentrations of LF(N) promoted assembly of the prepore from proteolytically activated PA, whereas high concentrations inhibited assembly of both the prepore and the ternary complex. A self-assembly scheme of anthrax toxin complexes is proposed.

Antigens, Bacterial↗
PubMed2005

Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.

Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.

Animals↗
PubMed2005

Whole-cell voltage clamp measurements of anthrax toxin pore current.

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.

Animals↗
PubMed2005

A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore.

The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.

Amino Acid Sequence↗
PubMed2005

Interaction of the 20 kDa and 63 kDa fragments of anthrax protective antigen: kinetics and thermodynamics.

The action of anthrax toxin begins when the protective antigen (PA(83), 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA(20) (20 kDa) and PA(63) (63 kDa). After PA(20) dissociates, receptor-bound PA(63) spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol. Treatment of PA(83) with trypsin yielded PA(63) and a form of PA(20) lacking unstructured regions at the N- and C-termini. We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution. We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, k(off), of 3.3 x 10(-)(2) s(-)(1) (t(1/2) of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (k(on) = 1.4 x 10(5) M(-)(1) s(-)(1)) was followed by a slower unimolecular step (k = 3.5 x 10(-)(3) s(-)(1)) with an equilibrium isomerization constant, K(iso), of 2.1. The two-step mechanism most consistent with the data is one in which the dissociation of the PA(20).PA(63) complex is followed by an isomerization in the PA(63) moiety. Our results indicate that, following the cleavage of PA on the cell surface, PA(20) is largely dissociated within a minute. A slow isomerization step in PA(63) may then potentiate it for oligomerization and subsequent steps in toxin action.

Amino Acid Sequence↗
PubMed2004

Differences in the folding transition state of ubiquitin indicated by phi and psi analyses.

We compare the folding transition state (TS) of ubiquitin previously identified by using psi analysis to that determined by using analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from psi analysis has an extensive native-like chain topology, with a four-stranded beta-sheet network and a portion of the major helix. According to analysis, however, the TS is much smaller and more polarized, with only a local helix/hairpin motif. We find that structured regions can have values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.

Computer Simulation↗
PubMed2004

Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin.

The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.

Acids↗
PubMed2004

Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously.

The 83 kDa protective antigen (PA(83)) component of anthrax toxin, after proteolytic activation, self-associates to form ring-shaped heptamers ([PA(63)](7)) that bind and aid delivery of the Edema Factor (EF) and Lethal Factor (LF) components to the cytosol. Here we show using fluorescence (Förster) resonance energy transfer that a molecule of [PA(63)](7) can bind EF and LF simultaneously. We labeled EF and LF with an appropriate donor/acceptor pair and found quenching of the donor and an increase in sensitized emission of the acceptor when, and only when, a mixture of the labeled proteins was combined with [PA(63)](7). Addition of unlabeled PA(63)-binding domain of LF to the mixture competitively displaced labeled EF and LF, causing a loss of energy transfer. In view of the known maximum occupancy of 3 ligand molecules per [PA(63)](7), these findings indicate that PA, EF, and LF can form mixtures of liganded toxin complexes containing both EF and LF.

Antigens, Bacterial↗
PubMed2004

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen.

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.

Antigens, Bacterial↗
PubMed2004

Discerning the structure and energy of multiple transition states in protein folding using psi-analysis.

We quantify the degree to which folding occurs along a complex landscape with structurally distinct pathways using psi-analysis in combination with a protein engineering method that identifies native, non-covalent polypeptide interactions and their relative populations at the rate-limiting step. By probing the proximity of two specific partners, this method is extremely well-suited for comparison to theoretical simulations. Using ubiquitin as a model system, we detect individual pathways with site-resolved resolution, demonstrating that the protein folds through a native-like transition state ensemble with a common nucleus that contains heterogeneous features on its periphery. The consensus transition state topology has part of the major helix docked against four properly aligned beta-strands. However, structural heterogeneity exists in the transition state ensemble, wherein peripheral regions are differentially populated according to their relative stability. Pathway diversity reflects the variable order of formation of these peripheral elements, which radiate outward from the common nucleus. These results, which show only moderate agreement with traditional mutational phi-analysis, provide an extraordinarily detailed and quantitative description of protein folding.

Amino Acid Sequence↗
PubMed2002

Contribution of hydrogen bonding to protein stability estimated from isotope effects.

An unresolved issue in structural biology concerns the relative contribution of H bonds to protein stability. We use the small molecules 4-acetamidobenzoic acid and N-acetylanthranilic acid as model compounds to relate the energetic contribution from hydrogen bonds (H bonds) to the deuterium/hydrogen amide isotope effect. N-Acetylanthranilic acid models carbonyl-amide H bonds formed during protein folding; 4-acetamidobenzoic acid models the unfolded state in which the amide H bonds to water. NMR is used to measure shifts in the pK(a) of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated. From the pK(a) shift, we obtain a quantitative scale factor: SF = partial partial differential(DeltaG(HB))/partial partial differential(RT ln Phi), where DeltaG(HB) is the change in free energy of an H bond upon isotope substitution and Phi is the fractionation factor. Isotope effect data also are reported for a small globular protein, lambda repressor, using the "C(m) experiment". The protein's isotope effect, which reports on the shape of the energy well, is converted to H-bonding free energy by applying the scale factor. We estimate that amide-related H bonds (amide-carbonyl and amide-water) contribute favorably to protein stability by approximately 30-50 kcal/mol in lambda repressor, GCN4 coiled coil, and cytochrome c but unfavorably by approximately 6 kcal/mol in ubiquitin. The results indicate that H-bond strength varies from one protein to another and presumably at different sites within the same protein.

Amides↗
PubMed2002

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.

Acid Anhydride Hydrolases↗