Dominant negative inhibition by fragments of a monomeric enzyme

Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.

Staphylococcal protein A: Unfolding pathways, unfolded states, and differences between the B and E domains

We present multiple native and denaturation simulations of the B and E domains of the three-helix bundle protein A, totaling 60 ns. The C-terminal helix (H3) consistently denatures later than either of the other two helices and contains residual helical structure in the denatured state. These results are consistent with experiments suggesting that the isolated H3 fragment is more stable than H1 and H2 and that H3 forms early in folding. Interestingly, the denatured state of the B domain is much more compact than that of the E domain. This sequence-dependent effect on the dimensions of the denatured state and the lack of correlation with structure suggest that the radius of gyration can be a misleading reaction coordinate for unfolding/folding. Various unfolding and refolding events are observed in the denaturation simulations. In some cases, the transitions are facilitated through interactions with other portions of the protein—contact-assisted helix formation. In the native simulations, the E domain is very stable: after 6 ns, the Cα root-mean-square deviation from the starting structure is less than 1.4 Å. In contrast, the native state of the B domain deviates more and its inter-helical angles fluctuate. In apparent contrast, we note that the B domain is thermodynamically more stable than the E domain. The simulations suggest that the increased stability of the B domain may be due to heightened mobility, and therefore entropy, in the native state and decreased mobility/entropy in the more compact denatured state.

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

A quantitative, high-throughput screen for protein stability

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.

Destabilization of Ca2+-free gelsolin may not be responsible for proteolysis in Familial Amyloidosis of Finnish Type

Mutations at position 187 in secreted gelsolin enable aberrant proteolysis at the 172–173 and 243–244 amide bonds, affording the 71-residue amyloidogenic peptide deposited in Familial Amyloidosis of Finnish Type (FAF). Thermodynamic comparisons of two different domain 2 constructs were carried out to study possible effects of the mutations on proteolytic susceptibility. In the construct we consider to be most representative of domain 2 in the context of the full-length protein (134–266), the D187N FAF variant is slightly destabilized relative to wild type (WT) under the conditions of urea denaturation, but exhibits a Tm identical to WT. The D187Y variant is less stable to intermediate urea concentrations and exhibits a Tm that is estimated to be ≈5°C lower than WT (pH 7.4, Ca2+-free). Although the thermodynamic data indicate that the FAF mutations may slightly destabilize domain 2, these changes are probably not sufficient to shift the native to denatured state equilibrium enough to enable the proteolysis leading to FAF. Biophysical data indicate that these two FAF variants may have different native state structures and possibly different pathways of amyloidosis.

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15–50°C, the S-form the second and the T-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.

Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17− deletion mutant compared with the isogenic wild-type hsp17+ revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a “membrane stabilizing factor” as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1′→4′ linkage 4a (oligomer I) or 6′→4′ linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)14 with a slightly reduced Tm value of 36.6°C, relative to 38.2°C for the control duplex d(T)14/d(A)14, but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)14 showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I the C6′ hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.

Characterization of DNA-Binding Proteins from Pea Mitochondria1

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.

Communication between noncontacting macromolecules

We present a quantitative experimental demonstration of solvent-mediated communication between noncontacting biopolymers. We show that changes in the activity of a solvent component brought about by a conformational change in one biopolymer can result in changes in the physical properties of a second noncontacting biopolymer present in solution. Specifically, we show that the release of protons on denaturation of a donor polymer (in this case, a four-stranded DNA tetraplex, iDNA) modulates the melting temperature of a noncontacting, acceptor polymer [in this case poly(A)]. In addition to such proton-mediated cross talk, we also demonstrate counterion-mediated cross talk between noncontacting biopolymers. Specifically, we show that counterion association/release on denaturation of native salmon sperm DNA (the donor polymer) can modulate the melting temperature of poly(dA)⋅poly(dT) (the acceptor polymer). Taken together, these two examples demonstrate how poly(A) and poly(dA)⋅poly(dT) can serve as molecular probes that report the pH and free salt concentrations in solution, respectively. Further, we demonstrate how such through-solvent dialogue between biopolymers that do not directly interact can be used to evaluate (in a model-free manner) association/dissociation reactions of solvent components (e.g., protons, sodium cations) with one of the two biopolymers. We propose that such through-solution dialogue is a general property of all biopolymers. As a result, such solvent-mediated cross talk should be considered when assessing reactions of multicomponent systems such as those that exist in essentially all biological processes.

Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu.

Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex.

cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2[d(GpG)-N7(1), -N7(2)]] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by approximately 9 degrees C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25 degrees C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslink-induced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslink-induced perturbations might modulate protein binding.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.