Proteasome-cytochrome c interactions: a model system for investigation of proteasome host-guest interactions

Owing to its high thermal stability and structural simplicity, the archaebacterium Thermoplasma Acidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzyme complex consists of a barrel-shaped 20S core (approximately 700kDa) comprised of four stacked seven-membered rings with a alpha(7)beta(7)beta(7)alpha(7) subunit structure situated around a 7-fold symmetry axis. The hollow interior of the proteasome has three large interconnected chambers with narrow (13 A diameter) entrances from solution located at either end of the barrel. The 14 beta-subunit proteolytic sites are located on the inner surface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cyt c) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditions of temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c is extensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasome digest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached heme prosthetic group are translocated to the central chamber where proteolysis occurs. In the presence of site-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequestered inside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis of the complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cyt c is essentially intact within the inhibited proteasome. High-performance liquid chromatography data show that (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can be formed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt c occurs.     

Using cyclic peptide mixtures as probes for metal ion host-guest interactions

Summary Mixtures of cyclic peptides, formed by head-to-tail cyclizations of side-chain resin-bound linear sequences, have been prepared using solid-phase synthesis. Fast atom bombardment mass spectrometry of cyclic peptides with various metal ions can reveal preferred modes of host-guest patterns, albeit in a nonquantitative manner. This approach could prove useful for more rapid screening of potential peptide ionophores. A cyclic heptapeptide with a dipeptide tail proved to be a particularly effective host for a Ca²⁺ ion; in a small three-component mixture, cyclo[Gly-Asp-d-Pro-Xxx-Asp-d-Pro-Asp(Aca-Phe-NH₂)], binding to Ca²⁺ varied from Xxx=N-MeAla>GlySar. In a 15-component mixture, cyclo[Pro-Xxx-Asn-Pro-Xxx-Asn] where Xxx=Ala, Glu, Leu, Lys or Phe, there were no significant differences with respect to binding to metal ions. We believe this to be the first reported use of cyclic peptide libraries for screening metal ions to discern host-guest relationships.Abbreviations Aca aminocaproic acid - Boc tert-butyloxycarbonyl - BOP benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate - DCM dichloromethane - DIEA diisopropylethylamine - DMF N,N-dimethylformamide - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - pMBHA 4-methylbenzhydrylamine - TFA trifluoroacetic acid This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Specific interactions in modified chitosan systems

This paper concerns the bulk and interfacial properties of a series of alkylated chitosans having different alkyl chain lengths grafted randomly along the main chitosan chain. Chitosan has a low degree of acetylation (5%); on chitosan derivatives, the role of the degree of grafting and of length of the alkyl chains are examined. The optimum alkyl chain length is C12 and the degree of grafting 5% to get physical gelation based on the formation of hydrophobic domains. The cross-linking is essentially controlled by the salt concentration: it is shown that 0.025 M AcONa is needed to screen electrostatic interchain repulsions. Hydrophobic interactions produce highly non-Newtonian behavior with large thinning behavior; this behavior is suppressed in the presence of cyclodextrins able to cap the hydrophobic alkyl chains.The interfacial properties of the chitosan derivatives were tested for the air/aqueous solution interfaces. Specifically, the role of their structure on the kinetic of film formation was examined showing that excess of external salt favors the stabilization of the interfacial film. The derivatives with a higher degree of substitution and longer alkyl chains are more efficient and give a higher elastic modulus compared to the model surfactant as a result of the chain properties.     

Development of cyclic AMP receptor protein-based artificial transcription factor for intensifying gene expression

Applied Microbiology and Biotechnology - Vector-dependent gene overexpression typically relies on an efficient operon and sufficient RNA polymerases (RNAPs). The lac (lactose) operon is a paradigm...     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

Specific antigen/antibody interactions measured by force microscopy.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.     

Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins.

One of the components of the RecA-LexA-controlled SOS response in Escherichia coli cells is an inducible error-prone DNA replication pathway that results in a substantial increase in the mutation rate. It is believed that error-prone DNA synthesis is performed by a multiprotein complex that is formed by UmuC, UmuD', RecA, and probably DNA polymerase III holoenzyme. It is postulated that the formation of such a complex requires specific interactions between these proteins. We have analyzed the specific protein-protein interactions between UmuC, UmuD, and UmuD' fusion proteins, using a Saccharomyces cerevisiae two-hybrid system. In agreement with previous in vitro data, we have shown that UmuD and UmuD' are able to form both homodimers (UmuD-UmuD and UmuD'-UmuD') and a heterodimer (UmuD-UmuD'). Our data show that UmuC fusion protein is capable of interacting exclusively with UmuD' and not with UmuD. Thus, posttranslational processing of UmuD into UmuD' is a critical step in SOS mutagenesis, enabling only the latter protein to interact with UmuC. Our data seem to indicate that the integrity of the entire UmuC sequence is essential for UmuC-UmuD' heterotypic interaction. Finally, in our studies, we used three different UmuC mutant proteins: UmuC25, UmuC36, and UmuC104. We have found that UmuC25 and UmuC36 are not capable of associating with UmuD'. In contrast, UmuC104 protein interacts with UmuD' protein with an efficiency identical to that of the wild-type protein. We postulate that UmuC104 protein might be defective in interaction with another, unknown protein essential for the SOS mutagenesis pathway.     

Hydrophobicity-induced pK shifts in elastin protein-based polymers.

Three polypentapeptides--poly[0.8(GVGVP), 0.2(GEGVP)], poly[0.8(GVGIP), 0.2(GEGIP)], and poly[0.75(GFGVP), 0.25(GEGVP)]--all analogues of the polypentapeptide of elastin--(Val1-Pro2-Gly3-Val4-Gly5)n or poly(VPGVG)--have been prepared to determine the effect of changing the hydrophobicity, i.e., Val1----Ile1 (I) and Val4----Phe4 (F), on the pKa and the temperature dependence of pKa of the Glu (E) residue. Shifts in pKa as large as 1.7 units are observed and the temperature dependence is much steeper for the structure-dependent proximity of the more hydrophobic Ile1 residues to the Glu4 residue. Even though this system is dominated by the inverse temperature transition of hydrophobically driven folding on raising the temperature, the effect of adding 0.15 N NaCl is to suppress the hydrophobicity-induced pKa shift.     

Specific hyposensitization of microbial allergy by using a synthetic artificial polyelectrolyte

In this work the effectiveness of complex immunotherapy, including specific inhalation hyposensitization with the introduction of artificial synthetic polyelectrolite, was studied. Specific allergen produced a good effect (75%) in the inhalation hyposensitization of hemophilic allergy. The intramuscular injection of artificial synthetic polyelectrolite, made in addition to the inhalation of allergen, produced a better desensitizing effect than the separate administration of allergen or polyelectrolite. The multiple administration of polyelectrolite produced a desensitizing effect on allergic reactions of type I with the tendency towards the decrease of reactions of type IV. Good prospects for the development of methods for special treatment with homologous allergen in combination with NA-5 in cases of microbial sensitization under the control of immunocompetent cells were shown.     

Towards the design of host-guest complexes: biotin and urea derivatives versus artificial receptors

Molecular mechanics calculations (Macromodel v.5.0 and v.8.1) have been used in order to correlate the minimized energies of the complexes with the binding constant Kb values measured on two hosts and five urea derivatives including methyl biotin. Kb values obtained by means of NMR titrations, in the right concentration range between 20 and 80% of saturation, correlate well with the energies provided by the molecular modeling study of the complexes.     

Specific interactions in proteins due to proton fluctuations

A theory of site–site interaction due to proton/charge fluctuations in protein molecules has been developed. It is shown that, with the proper geometric configuration of identical ionizable groups on matching sites, a specific attraction may be established. This attractive force has a bell-shaped pH dependence and is maximal close to the pK of the groups involved. Various types of protein interactions are examined in the light of this theory.     

Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes.

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.     

Murine transfer factor. III. Specific interactions between transfer factor and antigen

The interactions between dialyzable transfer factor and antigens have been studied. Incubation of transfer factor-containing dialysates from ferritin-sensitized mice or ferritin-coated plastic surfaces removed the antigen-sensitizing activity; incubations of the same preparations on cytochrome c-coated surfaces did not. Similar results were obtained when cytochrome c-transfer factor was studied. Incubation on cytochrome c-coated surfaces removed the activity, but incubation on ferritin-coated surfaces did not. Specific transfer factor activities could be recovered by elution with 8 M urea or acetonitrile. The finding of interactions between transfer factor and antigens provides evidence for a molecular basis of the specificity of the immunologic effects of transfer factor. This technique may also enable us to obtain amounts of specific material that are adequate for chemical analysis.     

Specific interactions versus counterion condensation. 1. Nongelling ions/polyuronate systems

The characteristics of the interaction between nongelling divalent cations (typically Mg(2+)) and polyuronates have been explored by means of isothermal calorimetry. In particular, three polyuronates mimicking separately guluronan (polyguluronate, polyG), mannuronan (polymannuronate, polyM), and polyalternating (polyMG), the three block-components of natural alginate samples, have been treated with divalent ions, and the enthalpy of mixing was determined for different values of the [M(2+)]/[Polym](rep.unit) ratio. Despite the absence of a site-specific chemical bonding between the two, as confirmed by circular dichroism spectroscopy, a substantial deviation of the experimental enthalpy of mixing from the theoretical behavior, as predicted by the classical counterion condensation (CC) theory, was observed. Such deviation has been interpreted in terms of a "generic" nonbonding affinity of the condensed divalent counterion for the polyelectrolytes. The mathematical formalism of the CC theory was extended to include a contribution to the (reduced) free energy and enthalpy arising from the counterion affinity, g(aff,0) and h(aff,0), and allowed the parametrical calculation of the fraction of divalent counterions condensed as function of the reduced thermodynamic quantity g(aff,0). A best fit procedure of the experimental enthalpy of mixing allowed the g(aff,0) and h(aff,0) pair to be estimated for each of the different polyuronates considered, revealing differences in the three samples. In qualitative terms, the results obtained seem to suggest a notable contribution of the desolvation process (i.e., release of structured water as a consequence of the interaction between the divalent counterion and the uronate group) to the enthalpy of affinity for polyM which is counterbalanced and overcome by an ion pairing term (i.e., partial formation of ion-ion and/or ion-dipole bonds) for polyG and polyMG, respectively.     

Synthesis of a cationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase

An N-methylated, cationic pyridoxamine conjugation reagent was synthesized and tethered via a disulfide bond to a cysteine residue inside the cavity of intestinal fatty acid binding protein. The conjugate was characterized and the kinetic parameters compared to its nonmethylated pyridoxamine analogue. Kinetic isotope effects were used for further mechanistic analysis. Taken together, these experiments suggest that a step distinct from deprotonation of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal energetics of reactions catalyzed by the conjugate containing the N-methylated cofactor appear to be different suggesting that the MPx reagent will be useful in future experiments designed to alter the catalytic properties of semisynthetic transaminases.     

A computational study of a host-guest complex

The geometry and energetics of a complex involving pyrazine and an acridine diacid cleft-like host designed by Rebek were investigated at several levels of theory. Molecular mechanics (using the Tripos and CHARMm force fields), semiempirical quantum chemical approaches (with the AM1 and PM3 methods), and an ab initio quantum chemical method (RHF/STO-3G) were used in the complete relaxation of the complex. The geometry of the complex optimized by the RHF/STO-3G method is in excellent agreement with a published X-ray structure; upon superposition, the rms deviation between the corresponding cleft heavy atoms is only 0.17 Å and the pyrazine molecules are superimposable. In addition, ab initio quantum chemical techniques were used to study the complex when the cleft is modeled by a pair of acetic acid molecules. All the calculations presented herein support a two-point interaction mechanism. The similarities found in the results for the full complex and the truncated model are consistent with a purely structural role for the acridine linker of the host. © 1997 John Wiley & Sons, Ltd.