Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution

Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 A away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.     

The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.     

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3α-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 °C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3α-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.     

pH effects on the stability and dimerization of procaspase-3

pH-dependent conformational changes are known to occur in dimeric procaspase-3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase-3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH-dependent dissociation below pH 5, so that the protein is mostly monomeric at pH 4. Consistent with this, the dimer unfolds via a four-state process between pH 8.5 and pH 4.75, in which the native dimer isomerizes to a dimeric intermediate, and the dimeric intermediate dissociates to a monomer, which then unfolds. In contrast, a small protein concentration dependence was observed by circular dichroism, but not by fluorescence emission, at pH 4.5 and pH 4.2. There was no protein-concentration dependence to the data collected at pH 4. Overall, the results are consistent with the redistribution of the population of native dimer (N(2)) to dimeric intermediate (I(2)) to monomeric intermediate (I), as the pH is lowered so that at pH 4, the "native" ensemble resembles the monomeric intermediate (I) observed during unfolding at higher pH. An emerging picture of the monomeric procaspase is discussed. Procaspase-3 is most stable at pH approximately 7 (24-26 kcal/mol), and while the stability decreased with pH, it was observed that dimerization contributes the majority (>70%) of the conformational free energy.     

Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.     

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation,dimerization and EGF hormone binding

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N‐glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF–EGFR binding takes place through a large‐scale induced‐fitting mechanism. Proteins 2017; 85:561–570. © 2016 Wiley Periodicals, Inc.     

Cadherin conformations associated with dimerization and adhesion

To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.     

Crystal structure of the biglycan dimer and evidence that dimerization is essential for folding and stability of class I small leucine-rich repeat proteoglycans

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-angstroms resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a T(m) of 45-46 degrees C but refolds completely upon cooling to 25 degrees C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel beta sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.     

A functionally active dimer of Mycobacterium tuberculosis Malate synthase G

Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The pleotropic function of this protein by virtue of its intracellular/extracellular localization and its behavior as an adhesin and virulence factor is quite enigmatic. Despite its importance in mycobacterium persistence, we do not know much about its biophysical and biochemical properties. Earlier reports suggest that the enzyme exists only as a monomer in prokaryotes; however, we observed the existence of both active monomer and dimer forms of the enzyme under physiological conditions. The dimeric form of the enzymes is more stable as compared to the monomeric form as evident from various biophysical parameters. In addition, the dimeric enzyme also shows enhanced stability against proteolysis than the monomers. Based on these studies, it seems that dimerization is an important factor in regulating stability. The differential localization and diverse functions of malate synthase other than its enzymatic role might be triggering the stabilization of the enzyme dimer and modulation of activity and stability in vivo.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.     

The dimerization of folded monomers of ribulose 1,5-bisphosphate carboxylase/oxygenase

Spontaneous refolding and reconstitution processes of dimeric ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum have been investigated using size-exclusion high performance liquid chromatography (HPLC), spectroscopic, and activity measurements. When the unfolded Rubisco in guanidine hydrochloride is diluted at 4 degrees C, a folding intermediate (Rubisco-I) is rapidly formed, which remains in an unstable monomeric state and gradually develops into folded monomer (Rubisco-M) at 4 degrees C but undergoes irreversible aggregation at 25 degrees C. Refolding of Rubisco-I to Rubisco-M is a very slow process, taking about 20 h for 70% conversion at 4 degrees C. Rubisco-M is stable at 4 degrees C and is capable of forming an active dimer spontaneously when incubated at a temperature higher than 10 degrees C. The dynamic dimerization process has been measured in a temperature range of 4-35 degrees C by HPLC, and the results demonstrate that the dimerization is strongly facilitated by the temperature. It is found that dithiothreitol is essential for the spontaneous reconstitution of Rubisco.     

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.     

The catalase activity of ferrihaems

1. The variation of the specific stoicheiometric catalatic activity of proto- and deuteroferrihaem with total ferrihaem concentration has been studied at 25 degrees C over a wide range of pH. For deuteroferrihaem the results imply that only monomeric ferrihaem species contribute significantly to the catalatic activity. Protoferrihaem is more highly dimerized in solution and, in this system, contributions to the catalatic activity from both monomeric and dimeric ferrihaem species were observed. The ratio of the specific activity of protoferrihaem monomer to that of dimer varied from approximately 20 at pH7 to 5x10(4) at pH12.2. 2. The specific activity of protoferrihaem monomer closely resembles that of deuteroferrihaem monomer, both in magnitude and pH-dependence. In both cases the activity is inversely proportional to [H(+)]. In contrast, the activity of catalase is independent of pH in the range 5-10. At pH13 the activity of ferrihaem monomer becomes equal to the maximal activity of catalase. The results are in good agreement with those reported by Brown et al. (1970b) and provide support for the assumptions upon which this previous analysis relied. 3. Information from the literature concerning the catalatic activity and dimerization of the iron(III) complex of 4,4',4',4'-tetrasulphophthalocyanine (Waldmeier & Sigel, 1971; Sigel et al., 1971) have been re-analysed. The results imply that both the monomeric and dimeric complexes contribute to catalatic activity and these activities closely resemble those of the corresponding protoferrihaem species.     

Insights into the regulatory mechanism for caspase-8 activation

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, we compared active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8). We demonstrate that wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The K(D) for dimeric wtC8 is approximately 50 micro M and decreases when inhibitor bound. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. Our findings suggest that dimerization is the crucial factor for caspase-8 activation.     

Conformational change coupling the dimerization and activation of KSHV protease

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.     

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.     

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.