ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase.

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.

Variability in the role of the gasbladder in fish audition.

The teleost gasbladder is believed to aid in fish audition by transferring pressure components of incoming sound to the inner ears. This idea is primarily based on both anatomical observations of the mechanical connection between the gasbladder and the ear, followed by physiological experiments by various researchers. The gasbladder movement has been modeled mathematically as a pulsating bubble. This study is extending the previous work on fish with a physical coupling of the gasbladder and ear by investigating hearing in two species (the blue gourami Trichogaster trichopterus, and the oyster toadfish Opsanus tau) without a mechanical linkage. An otophysan specialist (the goldfish Carassius auratus) with mechanical coupling, is used as the control. Audiograms were obtained with acoustically evoked potentials (e.g., auditory brainstem response) from intact fish and from the same individuals with their gasbladders deflated. In blue gourami and oyster toadfish, removal of gas did not significantly change thresholds, and evoked potentials had similar waveforms. In goldfish thresholds increased by 33-55 dB (frequency dependent) after deflation, and major changes in evoked potentials were observed. These results suggest that the gasbladder may not serve an auditory enhancement function in teleost fishes that lack mechanical coupling between the gasbladder and the inner ear.

Identification of the predominant non-native histidine ligand in unfolded cytochrome c.

The heme and its two axial ligands, His18 and Met80, play a central role in the folding/unfolding mechanism of cytochrome c. Because of the covalent heme attachment, His18 remains bound under typical denaturing conditions, while the more labile Met80 ligand is replaced by an alternate histidine ligand. To distinguish between the two possible non-native histidine ligands in horse cytochrome c, variants with a His26 to Gln or His33 to Asn substitution were prepared using a yeast expression system. Protonation of the non-native histidine ligand in the GuHCl-denatured state results in a pronounced blue shift of the Soret heme absorbance band (low-spin to high-spin transition). While substitution of His26 has no effect on the apparent pKa of this transition (5.7 +/- 0.05), the H33N variant exhibits a substantially higher pKa (6.1 +/- 0.05), indicating that His33 is the dominant sixth heme ligand in denatured cytochrome c and that His26 (or another nitrogenous group) acts as a ligand in the absence of a histidine at position 33. The kinetics of the pH-induced ligand dissociation shows two phases which were assigned to each of the two histidine ligands on the basis of their distinct temperature dependence. Despite their nearly identical equilibrium unfolding transitions, the two histidine mutants show differences in their folding kinetics. While the kinetic behavior of H26Q cyt c is very similar to that of the wild-type, the H33N mutation leads to loss of a kinetic phase with a rate in the 2-10 s-1 range that has previously been attributed to the rate-limiting dissociation of a trapped non-native histidine, which is thus identified as His33.

Kinetic role of early intermediates in protein folding.

The traditional view that partly folded intermediates are important for directing a protein toward the native state has been challenged by the notion that proteins can intrinsically fold rapidly in a single step if kinetic complications due to slow conformational events are avoided. Intermediates that accumulate within the first few milliseconds of folding are, however, a common observation even for small single-domain proteins. Recent spectroscopic studies, coupled with quantitative kinetic analysis, suggest that folding is facilitated by the rapid formation of compact intermediates with some native-like structural features.

Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid.

Human transthyretin (TTR) can be transformed into amyloid fibrils by partial acid denaturation to yield a monomeric amyloidogenic intermediate that self-associates into amyloid through quaternary structural intermediates, which are identified by sedimentation velocity methods. The monomeric amyloidogenic intermediate has substantial beta-sheet structure with a nonnative but intact tertiary structure as discerned from spectroscopic methods. Proteolysis sensitivity studies suggest that the C-strand-loop-D-strand portion of TTR becomes disordered and moves away from the core of the beta-sandwich fold upon formation of the monomeric amyloidogenic intermediate over the pH range 5.1-3.9. The single site mutations that are associated with early onset amyloid disease [familial amyloid polyneuropathy (FAP)] function by destabilizing tetrameric TTR. Under mild denaturing conditions, the FAP variants populate the monomeric amyloidogenic intermediate conformation, which assembles into amyloid, whereas wild-type TTR remains tetrameric and nonamyloidogenic. The FAP mutations do not significantly alter the native folded structure; instead, they appear to act by making the thermodynamics and perhaps the kinetics more favorable for formation of the amyloidogenic intermediate. Suppressor mutations have also been characterized that strongly stabilize tetrameric TTR and disfavor the formation of the monomeric amyloidogenic intermediate, thus inhibiting amyloid formation. The mechanistic details characterizing transthyretin amyloid fibril formation available from the biophysical studies outlined within have been utilized to develop a new therapeutic strategy for intervention in human amyloid disease. This approach features small molecules that bind with high affinity to the normal fold of transthyretin, inhibiting the quaternary and tertiary structural changes associated with the formation of the monomeric amyloidogenic intermediate that self-assembles into amyloid. Ligand binding to TTR stabilizes the native tetrameric fold, which is nonamyloidogenic.

Kinetic intermediates in the formation of the cytochrome c molten globule.

The relationship between molten globules and transient intermediates in protein folding has been explored by equilibrium and kinetic analysis of the compact acid-denatured A-state of cytochrome c. The chloride-induced formation of the A-state is a complex reaction with structural intermediates resembling those found under native refolding conditions, including a rapidly formed compact state and a subsequent intermediate with interacting N- and C-terminal helices. Together with mutational evidence for specific helix-helix packing interactions, this shows that the A-state is a stable analogue of a late folding intermediate. The L94A mutation blocks all folding steps after the initial collapse and its equilibrium state resembles early kinetic intermediates.

The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid.

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.

Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding.

The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (INC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (IC) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of INC) consistent with a strong decrease in the population of INC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of INC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of IC, the subsequent partially folded intermediate, INC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha-helices.

FAP mutations destabilize transthyretin facilitating conformational changes required for amyloid formation.

Functional transthyretin (TTR) can be transformed into amyloid by partial acid denaturation yielding a monomeric amyloidogenic intermediate which self-associates. The amyloidogenic intermediate has substantial beta-sheet structure with non-native but defined tertiary structure. pH-dependent proteolysis sensitivity studies have identified portions of TTR which become disordered and solvent-exposed in the amyloidogenic intermediate. These include the C-strand-loop D-strand portion of TTR which moves away from the core of the beta-sandwich fold. Mutations that are associated with early onset-amyloid disease (familial amyloidotic polyneuropathy; FAP) function by destabilizing tetrameric TTR in favour of the monomeric amyloidogenic intermediate which has a rearranged C-strand-loop D-strand region. In most cases the FAP mutations do not significantly alter the native folded structure, but instead act on the denaturation pathway by a mechanism that is not completely understood. Interestingly, mutations have also been characterized which strongly stabilize tetrameric TTR and make amyloid formation very difficult at pHs accessible in vivo.

Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease.

The role that transthyretin (TTR) mutations play in the amyloid disease familial amyloid polyneuropathy (FAP) has been probed by comparing the biophysical properties of several TTR variants as a function of pH. We have previously demonstrated that the partial acid denaturation of TTR is sufficient to effect amyloid fibril formation by self-assembly of a denaturation intermediate which appears to be monomeric. Earlier studies on the most pathogenic FAP variant known, Leu-55-Pro, revealed that this variant is much less stable toward acid denaturation than wild-type TTR, apparently explaining why this variant can form amyloid fibrils under mildly acidic conditions where wild-type TTR remains nonamyloidogenic. The hypothesis that FAP mutations destabilize the TTR tetramer in favor of a monomeric amyloidogenic intermediate under lysosomal (acidic) conditions is further supported by the data described here. We compare the acid stability and amyloidogenicity of the most prevalent FAP variant, Val-30-Met, along with the double mutant, Val-30-Met/Thr-119-Met, which serves to model the effects of these mutations in heterozygous patients where the mutations are in different subunits. In addition, we have characterized the Thr-119-Met TTR variant, which is a common nonpathogenic variant in the Portuguese population, to further investigate the role that this mutation plays in protecting individuals who also carry the Val-30-Met mutation against the classically severe FAP pathology. This biophysical study demonstrates that Val-30-Met TTR is significantly less stable toward acid denaturation and more amyloidogenic than wild-type TTR, which in turn is less stable and more amyloidogenic than Thr-119-Met TTR. Interestingly, the double mutant Val-30-Met/Thr-119-Met is very similar to wild-type TTR in terms of its stability toward acid denaturation and its amyloidogenicity. The data suggest that the Thr-119-Met mutation confers decreased amyloidogenicity by stabilizing tetrameric TTR toward acid denaturation. In addition, fluorescence studies monitoring the acid-mediated denaturation pathways of several TTR variants reveal that the majority exhibit a plateau in the relative fluorescence intensity over the amyloid-forming pH range, i.e., ca. pH 4.3-3.3. This intensity plateau suggests that the amyloidogenic intermediate(s) is (are) being observed over this pH range. The Thr-119-Met variant does not exhibit this plateau presumably because the amyloidogenic intermediate(s) do(es) not build up in concentration in this variant. The intermediate is undoubtedly forming in the Thr-119-Met variant, as it will form amyloid fibrils at high concentrations; however, the intermediate is only present at a low steady-state concentration which makes it difficult to detect.

Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity.

A recently reported variant of human transthyretin (TTR), Leu-55-Pro, implicated as the causative agent in early-onset familial amyloid polyneuropathy was expressed and characterized, and its denaturation pathway and amyloidogenicity were compared to those of wild-type transthyretin. The overlap-extension polymerase chain reaction (PCR) methodology was used to introduce the Leu-55-Pro mutation into the transthyretin DNA sequence and to construct a new expression system. The Leu-55-Pro variant of transthyretin was expressed with a leader sequence to ensure secretion into the periplasmic space of Escherichia coli. Transthyretin's resistance to sodium dodecyl sulfate- (SDS-) induced denaturation was utilized to measure the quaternary stability as a function of pH employing SDS-polyacrylamide gel electrophoresis (PAGE) in the presence and absence of an amyloid fibril inhibitor, Z 3-14. These studies reveal that the Leu-55-Pro TTR tetramer is significantly less stable than wild-type TTR. This is relevant because we have previously shown that the partial denaturation of transthyretin is sufficient to effect amyloid fibril formation from a denaturation intermediate which may be a structured monomer. The ability of Leu-55-Pro TTR to denature to the amyloidogenic intermediate at pHs where the wild-type protein is stable may explain the variant's propensity to form amyloid fibrils in vitro and in vivo where the wild-type protein remains stable and nonamyloidogenic. Congo red binding, polarized light microscopy, and electron microscopy confirm the characteristic structure of amyloid fibrils produced from Leu-55-Pro TTR in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.

Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)