Hyperthermal stability of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb), recent additions to the globin family, display a hexa-coordinated (bis-histidyl) heme in the absence of external ligands. Although these proteins have the classical globin fold they reveal a very high thermal stability with a melting temperature (Tm) of 100 degrees C for Ngb and 95 degrees C for Cygb. Moreover, flash photolysis experiments at high temperatures reveal that Ngb remains functional at 90 degrees C. Human Ngb may have a disulfide bond in the CD loop region; reduction of the disulfide bond increases the affinity of the iron atom for the distal (E7) histidine, and leads to a 3 degrees C increase in the T(m) for ferrous Ngb. A similar Tm is found for a mutant of human Ngb without cysteines. Apparently, the disulfide bond is not involved directly in protein stability, but may influence the stability indirectly because it modifies the affinity of the distal histidine. Mutation of the distal histidine leads to lower thermal stability, similar to that for other globins. Only globins with a high affinity of the distal histidine show the very high thermal stability, indicating that stable hexa-coordination is necessary for the enhanced thermal stability; the CD loop which contains the cysteines appears as a critical region in the neuroglobin thermal stability, because it may influence the affinity of the distal histidine.     

Calorimetric investigation of the domain structure of human complement Cl-s: reversible unfolding of the short consensus repeat units

Cl-s is a multidomain serine protease that participates in Ca2+-dependent protein-protein interactions with other subcomponents of Cl, the first component of human complement. Proteolytically derived fragments that retain some of the functional properties of the parent protein have been isolated, and their thermal stability has been investigated by differential scanning calorimetry. Three endothermic transitions are observed in whole Cl-s near 37, 49, and 60 degrees C in 0.05 M Tris-HCl, pH 7.2, containing 0.22 M NaCl and 0.1 mM EDTA. The first (37 degrees C) and third (60 degrees C) transitions are also seen in Cl-s-A, a derivative comprised mainly of the intact nonenzymatic A chain. The second (49 degrees C) and third transitions are seen in Cl-s-gamma B, a fragment comprised of the intact B chain, disulfide linked to the C-terminal gamma region of the A chain. Thus, the first transition, which is alone stabilized by Ca2+, corresponds to the melting of the N-terminal alpha beta region of the A chain, the second to the melting of the catalytic B chain domain, and the third to the gamma region. The gamma region is comprised of two homologous short consensus repeat (SCR) motifs that are also found in several other complement and coagulation proteins. A new 24-kDa fragment, Cl-s-gamma, which contains these two SCRs, was isolated from plasmic and chymotryptic digests of Cl-s-A. Cl-s-gamma exhibits a reversible transition near 60 degrees C corresponding to the highest temperature peak in whole Cl-s and Cl-s-A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.     

Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.     

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.     

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.     

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.     

Folding of collagen IV

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

A series of nucleobase-modified siRNA duplexes containing "rare" nucleosides, 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s(2)U or Psi units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DeltaTm 3.9 and 6.6 degrees C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s(2)U and Psi units at their 3'-ends and with a D unit at their 5'-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s(2)U and Psi units at their 5'-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 3'-adjacent to the mutation site. Thermally stable siRNA molecules containing several s(2)U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.     

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.     

Domains in lambda Cro repressor. A calorimetric study.

Thermodynamic properties of a mutant lambda Cro repressor with Cys replacing Val55 were studied calorimetrically. Formation of the S-S cross-link between neighboring Cys55 residues in this dimeric molecule leads to stabilization of a structure formed by the C-terminal parts of the two polypeptide chains, which behave as a single cooperative domain upon protein denaturation by heating. This composite domain is very stable at neutral pH and disrupts at 110 degrees C. The S-S-cross-linked tryptic fragment (residues 22-66), which includes this C-terminal domain, has similar stability. The N-terminal parts of the polypeptide chains do not form any stable structure when isolated, but in S-S-cross-linked dimer, they form a single cooperative block which melts in an all-or-none way 9 degrees C higher than the un-cross-linked protein. The observed cooperation of the distant N-terminal parts in dimer raises questions regarding lambda Cro repressor structure in solution.     

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.     

p53 contains large unstructured regions in its native state

The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.     

Stabilisation of the Fc fragment of human IgG1 by engineered intradomain disulfide bonds

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.     

Structure formation in the C terminus of type III collagen guides disulfide cross-linking

In type III collagen the main triple-helical domain is followed by a disulfide knot and the C-terminal propeptide, which are both essential for nucleation, stabilization and registration of the triple helix. We demonstrate that oxidative inter-chain disulfide bridging does not occur between the knot sequences GlyProCysCysGly of dissociated randomly coiled chains. N-terminal fusion of the obligatory trimeric domain of mini-fibritin is able to direct this process efficiently, demonstrating a folded precursor mechanism in which the thiol groups have to be properly placed for the formation of native disulfide bonds. The natural C-propeptide domain may act in a similar way as the mini-fibritin domain. After disulfide linkage and triple-helix formation the catalyzing mini-fibritin domain was removed by thrombin cleavage. In this way a short but stable triple-helical collagen fragment was expressed in Escherichia coli for structural and functional studies.     

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.