Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability

The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.     

Conservation of cysteine residues in fungal histidine acid phytases

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.     

Conservation of amino acids into multiple alignments involved in pairwise interactions in three-dimensional protein structures

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.     

Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236

Improvement of thermal stability of the Bacillus stearthermophilus No. 236 endo-beta-1,4-xylanase (XynA) was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using the disulfide bond design program (Disulfide by Designtrade mark) to identify residue pairs having the favorable geometric characteristics for disulfide formation. Subsequently, the selected 25 amino acid pairs were filtered with the evolutionarily conserved Cys residues identified by alignment of 34 family 11 mesophilic and thermophilic xylanases, and also by doing inspection of the molecular model of the xylanases. Only one pair (Ser100 and Asn150) was finally chosen, and the respective amino acids were substituted with cysteine residues. The newly designed disulfide bridge increased thermostability of the XynA about 5 degrees C. This improved thermal stability was supported by the increase in the energy barrier for inactivation. As expected, the mutant XynA SNC demonstrated its better use in the hydrolysis of xylan at substantially higher temperatures than permitted by its native counterpart. The mutation had little influence on the catalytic efficiency and other functional properties of the XynA. In conclusion, it is evident that the strategically placed disulfide bridge has made the XynA be more effective in resisting thermal inactivation.     

A unique lantibiotic, thermophilin 1277, containing a disulfide bridge and two thioether bridges

Aims:  To identify the chemical structure of a bacteriocin, thermophilin 1277, produced by Streptococcus thermophilus SBT1277.
Methods and Results:  Thermophilin 1277 was purified and partial N-terminal sequence analysis revealed 6 unidentified amino acids amongst 31 amino acids residues. A 2·7-kbp region containing the thermophilin 1277 structural gene ( tepA ) encoding 58 amino acids was cloned and sequenced. Mature thermophilin 1277 (33 amino acids) was preceded by a 25-amino acid putative leader peptide containing a double glycine cleavage motif. Peptide sequence analysis following chemical modification of thermophilin 1277 revealed that the Cys21 and Cys29 residues form a disulfide bridge and that Thr8 or Thr10 forms two 3-methyllanthionines with Cys13 or Cys32 via thioether bridges. Antimicrobial activity was disrupted by ethanethiol or reductive agent treatments, indicating that the internal amino acid modifications are crucial for the activity.
Conclusions:  Thermophilin 1277 from Strep. thermophilus SBT1277 belongs to the class of AII-type lantibiotics that has a disulfide and two thioether bridges.
Significance and Impact of the Study:  This is the first report of a lantibiotic produced by a GRAS species of Strep. thermophilus ; thermophilin 1277 has a unique structure containing both a disulfide bridge and two thioether bridges that are crucial for its activity.     

Thermodynamics and kinetics of protein folding: an evolutionary perspective

This article appeals to an evolutionary model which postulates that primordial proteins were described by small polypeptide chains which (i) lack disulfide bridges, and (ii) display slow folding rates with multi-state kinetics, to determine relations between structural properties of proteins and their folding kinetics. We parameterize the energy landscape of proteins in terms of thermodynamic activation variables. The model studies evolutionary changes in these thermodynamic parameters, and we invoke relations between these activation variables and structural properties of the protein to predict the following correspondence between protein structure and folding kinetics. 1. Proteins with inter- and intra-chain disulfide bridges: large variability in both folding rates and stability of intermediates, multi-state kinetics. 2. Proteins which lack inter and intra-chain disulfide bridges. 2.1 Single-domain chains: fast folding rates; unstable intermediates; two-state kinetics. 2.2 Multi-domain monomers: intermediate rates; metastable intermediates; multi-state kinetics. 2.3 Multi-domain oligomers: slow rates; metastable intermediates; multi-state kinetics. The evolutionary model thus provides a kinetic characterization of one important subfamily of proteins which we describe by the following properties: Folding dynamics of single-domain proteins which lack disulfide bridges are described by two-state kinetics. Folding rate of this class of proteins is positively correlated with the thermodynamic stability of the folded state.     

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.     

Sindbis virus glycoprotein E1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections

The E1 membrane glycoprotein of Sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (B. A. Abell and D. T. Brown, J. Virol. 67:5496-5501, 1993; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have examined the disulfide bonds in E1 and have determined that the E1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the protein into two domains at amino acid 129. These separate sets of disulfides may stabilize and define the structural and functional regions of the E1 protein.     

The role of salt bridges on the temperature adaptation of aqualysin I,a thermostable subtilisin-like proteinase

Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combining atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters.Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the properties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase.     

Disulfide bridges in interleukin-8 probed using non-natural disulfide analogues: dissociation of roles in structure from function.

The structural and functional roles of the two disulfide bridges in interleukin-8 (IL-8) were addressed using IL-8 analogues with covalently modified disulfide bridges. The analogues were prepared using chemical synthesis by replacement of a cysteine for either homocysteine, penicillamine, or selenocysteine and on folding resulted in a covalently modified disulfide. Deletion of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In contrast, all of the analogues with modified disulfide bridges had native tertiary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the functional effects of the changes to the disulfide. Modification to the disulfide bridge between cysteines 9 and 50 had only a modest effect on IL-8 function. In contrast, alterations to the 7-34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bridges are required for maintenance of the native tertiary fold, their role in determining IL-8 activity is distinct. We propose that 7-34 disulfide has a direct role in determining receptor binding and activation, whereas the 9-50 was not directly involved. The synthesis of non-natural disulfide analogues is a novel general approach to structure-activity relationships of disulfide bridges. The demonstration that the participation of disulfide bridges in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method will lead to increased understanding of the roles of disulfide bridges in proteins.     

Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors

We have altered the amino acid sequence of the lac repressor one residue at a time by utilizing a collection of nonsense suppressors that permit the insertion of 13 different amino acids in response to the amber (UAG) codon, as well as an additional amino acid in response to the UGA codon. We used this collection to suppress nonsense mutations at 141 positions in the lacI gene, which encodes the 360 amino acid long lac repressor, including 53 new nonsense mutations which we constructed by oligonucleotide-directed mutagenesis. This method has generated over 1600 single amino acid substitutions in the lac repressor. We have cataloged the effects of these replacements and have interpreted the results with the objective of gaining a better understanding of lac repressor structure, and protein structure in general. The DNA binding domain of the repressor, involving the amino-terminal 59 amino acids, is extremely sensitive to substitution, with 70% of the replacements resulting in the I- phenotype. However, the remaining 301 amino acid core of the repressor is strikingly tolerant of substitutions, with only 30% of the amino acids introduced causing the I- phenotype. This analysis reveals the location of sites in the protein involved in inducer binding, tighter binding to operator and thermal stability, and permits a virtual genetic image reconstruction of the lac repressor protein.     

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.     

Structural characterization of functionally important regions of the Escherichia coli heat-stable enterotoxin STIb

The biological properties of the Escherichia coli enterotoxin STIb (STA-3, STh) reside in a 13 amino acid C-terminal domain, abbreviated STIb(6-18). This tridecapeptide contains six cysteine residues involved in three intramolecular disulfide bridges. The solution structure of STIb(6-18) has been modeled as a series of three consecutive reverse turns [Gariépy et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 83, 8907-8911]. Synthetic tridecapeptide analogues of STIb(6-18) with single amino acid substitutions at non-cysteine sites, as well as a truncated decapeptide lacking one of the three disulfide bridges, were prepared in order to examine the relationship between primary sequence and biological activity. The relative affinity of each analogue for intestinal cell receptors only partially correlates with their dose-dependent ability to cause diarrhea in suckling mice, suggesting that subsaturation doses of the enterotoxin with respect to receptor occupancy on intestinal cells may be sufficient to cause diarrhea. Two substitutions in the central-turn region of the molecule, namely, Asn12----Ala and Ala14----D-Ala, resulted in a large decrease or loss of receptor binding activity as compared to native STIb(6-18), pointing out the functional importance of this region. Analogues containing replacements at other sites showed moderate to slight reductions in biological activity. In particular, residues in the C-terminal region appear to be less important for activity, although their presence remains essential, since a truncated analogue missing the last three amino acids is inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

Importance of the disulfide bridges in the antibacterial activity of human hepcidin

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.     

Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity.

Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.     

Total solution synthesis of human epidermal growth factor (h-EGF) by the assembly of nine building blocks.

Human epidermal growth factor (h-EGF) composed of 53 amino acids bearing three intramolecular disulfide bridges was synthesized by the maximum protecting solution method. The synthetic h-EGF coincided with recombinant h-EGF by reverse-phase HPLC, and the sites of three intramolecular disulfide bridges were ascertained by a thermolytic digestion. The synthetic h-EGF possessed m/z 6215.7 in its FAB-MS as expected, and exhibited compatible mitogenic activity.     

Total Solution Synthesis of Human Epidermal Growth Factor (h-EGF) by the Assembly of Nine Building Blocks

Human epidermal growth factor (h-EGF) composed of 53 amino acids bearing three intramolecular disulfide bridges was synthesized by the maximum protecting solution method. The synthetic h-EGF coincided with recombinant h-EGF by reverse-phase HPLC, and the sites of three intramolecular disulfide bridges were ascertained by a thermolytic digestion. The synthetic h-EGF possessed m/z 6215.7 in its FAB-MS as expected, and exhibited compatible mitogenic activity.