Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase.

Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.

Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods.

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.

Hydrophobic core malleability of a de novo designed three-helix bundle protein.

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.

Role of an alpha-helical bulge in the yeast heat shock transcription factor.

The heat shock transcription factor (HSF) is the master transcriptional regulator of the heat shock response. The identity of a majority of the genes controlled by HSF and the circumstances under which HSF becomes induced are known, but the details of the mechanism by which HSF is able to sense and respond to heat remains an enigma. For example, it is unclear whether HSF senses the heat shock directly or requires ancillary interactions from a heat-induced signaling pathway. We present the analysis of a series of mutations in an alpha-helical bulge in the DNA-binding domain of HSF. Deletion of residues in this bulged region increases the overall activity of the protein. Yeast containing the deletion mutant HSF are able to survive growth temperatures that are lethal to yeast containing wild-type HSF, and they are also constitutively thermotolerant. The increase in activity can be measured as an increase in both constitutive and induced transcriptional activity. The mutant proteins bind DNA more tightly than the wild-type protein does, but this is unlikely to account fully for the increase in transcriptional activity as yeast HSF is constitutively bound to its binding site in vivo. The stability of the mutant proteins to thermal denaturation is lower than wild-type, though their native-state structures are still well-folded. Therefore, the mutants may be structurally analogous to the heat-induced state of HSF, and suggest that the DNA-binding domain of HSF may be capable of sensing heat shock directly.

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Solution structure and dynamics of a de novo designed three-helix bundle protein.

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, alpha3D, is reported. The structure of alpha3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

The induction of respiratory allergy in guinea-pigs following intradermal injection of trimellitic anhydride: a comparison with the response to 2,4-dinitrochlorobenzene.

Guinea-pigs injected intradermally with the known respiratory sensitiser trimellitic anhydride (TMA) developed high-titre antigen-specific homocytotropic (IgG1 and IgE) antibodies. Many of the sensitised animals responded to a challenge by inhalation with either free TMA or a TMA-protein conjugate with a change in respiratory rate, reflecting the onset of bronchoconstriction. Guinea-pigs were also injected intradermally with 2,4-dinitrochlorobenzene (DNCB), which is a potent skin sensitiser in man but which has not been reported to cause respiratory allergy. These animals developed only low-titre homocytotropic antibodies and were unresponsive to an inhalation challenge with either free or conjugated hapten. The animals were, however, contact-sensitised to the chemical.

Sensitisation of guinea pigs by inhalation exposure to low molecular weight chemicals.

Guinea pigs could be immunologically sensitised (as shown by the development of antigen-specific homocytotropic antibodies) to toluene diisocyanate by exposing them for 3 h a day for 5 consecutive days to atmospheres containing free chemical. Pulmonary reactions could be elicited in many of the sensitised animals by challenging them with atmospheres containing protein conjugates of the chemical and then measuring changes in respiratory rate. Successful elicitation of pulmonary reactions appeared to depend upon a number of factors, including the quality of the protein conjugate used for the challenge, but possibly also the development of IgE as well as IgG1 antibodies. Antigen-specific homocytotropic antibodies were detected in guinea pigs similarly exposed by inhalation to two non-isocyanate respiratory allergens, trimellitic anhydride and a reactive dye. Although the animals were immunologically sensitised to the chemicals, challenge with atmospheres containing appropriate chemical-protein conjugates failed to stimulate changes in respiratory rate.

Control of the immune response to contact sensitizing chemicals by cutaneous antigen-presenting cells.

The fate of 2,4-dinitrochlorobenzene, a potent contact sensitizing chemical, and 2,4-dichloronitrobenzene, a non-sensitizer, was compared following their application to the skin of BALB/c mice. Although both chemicals were able to bind to protein in vitro and were capable of being absorbed across mouse skin in vivo, only 2,4-dinitrochlorobenzene was able to bind to cells in the skin and to induce the movement of these cells from the epidermis into the dermis and ultimately into the draining lymph nodes. The sensitization potential of a chemical may therefore be dependent on its ability to associate with and stimulate the efflux of cutaneous antigen-presenting cells.

Contact-sensitizing and tolerogenic properties of 2,4-dinitrothiocyanobenzene.

Epicutaneous application of 2,4-dinitrothiocyanobenzene (DNTB) is said to result in specific immunological hyporesponsiveness and fails to induce contact sensitization. However, we demonstrate that topical exposure to DNTB causes activation of the draining lymph node in mice and the induction of contact sensitization in both rodents and a single human volunteer. In mice and rats, pre-exposure to DNTB failed to impair subsequent responsiveness to the cross-reactive allergen 2,4-dinitrochlorobenzene. These data provide evidence that DNTB cannot be regarded as an exclusive tolerogen when applied epicutaneously and indicate that attempts to define the characteristics of tolerising chemicals from analysis of this agent may be misleading.

Enzyme linked immunosorbent assay (ELISA) for paraquat.

An Enzyme Linked Immunosorbent Assay (ELISA) has been developed for the estimation of paraquat. The amount of paraquat present in samples of human plasma was estimated in terms of the degree to which it combined with a rabbit antibody raised to a conjugate to paraquat with bovine serum albumin. The amount of residual, uncombined, antibody after being allowed to react with a conjugate of paraquat with keyhole-limpet haemocyanin bound to the surface of a polystyrene micro-titre plate, was estimated by the addition of an enzyme-labelled anti-rabbit IgG, followed after washing by addition of substrate and subsequent determination of optical density. Concentrations of paraquat in the range 0.3-10 ng/ml could be measured and the antibody showed a high degree of specificity. Results correlated well with those of a widely-validated radio-immunoassay but the ELISA was simpler and more sensitive.