Transverse Urea Gradient Gel Electrophoresis Research Articles

Plasma Proteolytic Cascade Activation during Neonatal Cardiopulmonary Bypass Surgery.

Neonates undergoing cardiopulmonary bypass (CPB) surgery to correct congenital heart defects often experience excessive bleeding. Exposure of blood to artificial materials during CPB may activate coagulation, complement and inflammatory pathways. In addition, the surgical stress placed on the haemostatic system may result in cross-activation of other plasma proteolytic cascades, which could further complicate physiological responses to the surgical procedure and post-operative recovery. Plasma protease inhibitors undergo distinct conformational changes upon interaction with proteases, and, thereby, can serve as endogenous biosensors to identify activation of the different proteolytic cascades. We tested the hypothesis that changes in the concentration and conformation of protease inhibitors regulating plasma proteolytic cascades during neonatal CPB are associated with post-operative bleeding. Plasma samples from 44 neonates were obtained at four time points across the surgical procedure. Anti-thrombin, antitrypsin, anti-chymotrypsin, anti-plasmin, C1-inhibitor and tissue factor pathway inhibitor (TFPI) concentrations and conformations were evaluated by enzyme-linked immunosorbent assay, transverse urea gradient gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The most striking changes were observed following heparin administration and were associated with the appearance of inactive forms of anti-thrombin and an increase in the plasma concentration of TFPI. Changes in anti-thrombin and TFPI remained evident throughout surgery and into the post-operative period but were not different between patients with or without post-operative bleeding. The concentration of antitrypsin decreased across surgery, but there was no significant accumulation of inactive conformations of any inhibitor besides anti-thrombin, indicating that widespread cross-activation of other plasma proteolytic cascades by coagulation proteases did not occur.

Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.

Role of lysine versus arginine in enzyme cold‐adaptation: Modifying lysine to homo‐arginine stabilizes the cold‐adapted α‐amylase from Pseudoalteramonas haloplanktis

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Delta65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H(4)B) and is the site for NO synthesis. The role of H(4)B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H(4)B) with the bacterially expressed Delta65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Delta65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Delta65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H(4)B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H(4)B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain.

The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.

Unfolding of hemoglobin variants—insights from urea gradient gel electrophoresis photon correlation spectroscopy and zeta potential measurements

The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke’s radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke’s radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke’s radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on native gels, but differing in their folding behavior.

Conformational Polymorphism of High MrGlutenin Subunits Detected by Transverse Urea Gradient Gel Electrophoresis

Unfolding behaviour of different allelic variants of high Mrglutenin subunits has been studied by transverse urea gradient gel electrophoresis, a system in which a gradient of urea (from 0 to 8 M) is generated perpendicularly to the direction of migration. A different unfolding pattern is observed between x- and y-type subunits. The former type of subunits generally present a broad unfolding pattern without any clear transition, as urea concentration increases, suggesting the existence of several conformational intermediates. On the contrary most of the y-type subunits present an unfolding pattern showing an abrupt decrease in mobility, with a single inflection point at the midpoint of the transition indicative of a protein unfolding in a single, cooperative, two-state transition. Native and bacterial expressed subunits, differing for the size of the repetitive domain, were used to assess the role of variation in size of such region on unfolding behaviour. Free energy values associated to unfolding process were calculated for most of the y-type subunits presenting a two state transition. In particular, different values were found for subunit Dy10 and Dy12, with the former being larger than the latter, suggesting a different stability of the two subunits. Possible implications for qualitative differences associated to allelic subunits are also discussed.

Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability.

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild-type and altered alkaline phosphatases show comparable catalytic behavior with k(cat) values between 51.3 and 69.5 s(-1) and Km values between 14.8 and 26.3 microM. The ultracentrifugation profiles indicate that the wild-type enzyme is more stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild-type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.

Structural and functional consequences of mutations within the hydrophobic cores of the HMG1-box domain of the Chironomus high-mobility-group protein 1a.

The high-mobility-group protein 1 box domain (HMG1-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three alpha-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trp14 of the Chironomus HMG1a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletion of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMG1a, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wild-type cHMG1a and [W14A]cHMG1a showed that the proteins unfold at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-8 M of the denaturant. Multiple contacts between the HMG1-BD and the DNA are probably responsible for the unusual stability of the complexes.

Structural and Functional Characteristics of Partially Disulfide-reduced Intermediates of Ovotransferrin N Lobe: CYSTINE LOCALIZATION BY INDIRECT END-LABELING APPROACH AND IMPLICATIONS FOR THE REDUCTION PATHWAY

Ovotransferrin N lobe contains six intrachain disulfides (SS-I/Cys10-Cys45; SS-II/Cys20-Cys36; SS-III/Cys115-Cys197; SS-IV/Cys160-Cys174; SS-V/Cys171-Cys182; SS-VI/Cys228-Cys242) in a single polypeptide chain of 332 amino acid residues. Upon the protein disulfide reduction with dithiothreitol under nondenaturing conditions, the intermediate species with four, three, and two disulfides were generated. The partially disulfide-reduced intermediates were isolated, and the localization of intact disulfides in the intermediates was determined by an indirect end-labeling method. This method included the S-cyanocysteine-specific protein fragmentation, followed by gel electrophoresis and the immunochemical visualization of the C terminus-intact fragments using antiserum raised against a non-cysteine C-terminal fragment (Ser280-Arg332). Results clearly showed that first SS-IV and SS-V, second SS-III, and then SS-VI are cleaved. No reduction was observed for SS-I and SS-II under the employed reducing conditions. The conclusion was confirmed by peptide mapping analyses for the same disulfide intermediates using reverse phase high performance liquid chromatography. Transverse urea gradient gel electrophoresis and visible absorption spectra revealed that the four-disulfide intermediate, but not the three- or two-disulfide intermediate, retains essentially the same iron-binding function as the native protein. By far-UV CD analyses, the residual native conformation of the partially disulfide-reduced intermediates was found to decrease with increased number of the reduced disulfides. Implications of the partially disulfide-reduced intermediates for the disulfide-reductive unfolding pathway in ovotransferrin N lobe are discussed.

Limited proteolysis of alpha 1-proteinase inhibitor (alpha 1-PI) in acute leukemia: studies on the resulting fragments and implication for the structure of the inactivated inhibitor.

In patients with acute myeloid leukemia, a 41 kDa glycoprotein appears in the urine during remission induction chemotherapy. We have recently reported on the isolation and preliminary characterization of this protein and the generation of specific monoclonal antibodies which showed that it is a proteolytically modified form of alpha 1-proteinase inhibitor (Dengler et al., 1992, Biol. Chem. Hoppe-Seyler 373, 581-588). In the paper presented here, results from further characterization experiments as well as from studies on the effects of proteolysis on the conformation and the resulting functional properties of the truncated inhibitor are reported. N-terminal amino acid sequence analysis showed that proteolysis has occurred in the N-terminal part as well as in the reactive site loop of alpha 1-PI. The resulting core protein of 41 kDa is composed of approximately 324 amino acid residues with the C-terminus located close to Lys343 of alpha 1-PI.A 4 kDa peptide remaining bound to this fragment throughout the entire purification procedure could be separated by SDS treatment. N- and C-terminal sequence analysis of this peptide after isolation by gel filtration showed that it is comprised of residues Ile359 up to Lys394, thus representing the peptide located C-terminal to the reactive site loop of alpha 1-PI. Transverse urea gradient gel electrophoresis indicates that the proteolyzed inhibitor is in the thermodynamically stable, relaxed (R)-conformation known for proteinase-complexed and cleaved serpins. The truncated inhibitor exhibits no chemotactic activity towards neutrophils when tested in a standard assay.

Folding patterns of immunoglobulin molecules identified by urea gradient electrophoresis.

The reversible denaturant-induced unfolding of immunoglobulin molecules has been analyzed by transverse urea gradient gel electrophoresis and the effects that urea-induced unfolding exerts on the functional properties associated with their variable region, i.e. antigen binding and idiotypic expression, have been determined by Western blot analysis. Results obtained from these experiments indicate that urea-induced unfolding of the immunoglobulin molecule is a highly cooperative reversible process that occurs through a two-state transition with no accumulation of intermediates. The unfolding transition has its midpoint at about 6.5 M urea and appears to be slow on the time scale of electrophoresis. Folding intermediates in rapid equilibrium with the unfolded state as well as molecular forms with different electrophoretic mobility can be detected during refolding reactions. Results from Western blot analysis confirm the highly cooperative reversible urea-induced unfolding of immunoglobulin molecules and demonstrate that the unfolding transition leads to disappearance of both antigen binding and idiotypic expression, whereas the ability to interact with antibodies directed to continuous epitopes of the variable region is preserved. After progressive removal of the denaturing agent, the variable region refolds into structures that regain the functional properties of the native conformation.

Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed and reactive site cleaved serpins: comparison of .alpha.1-proteinase inhibitor, .alpha.1-antichymotrypsin, antithrombin III, .alpha.2-antiplasmin, angiotensinogen, and ovalbumin

Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the proteinase-complexed form, a stable equimolar complex between the inhibitor and a target proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the proteinase component of the serpin-proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit proteinases in a manner similar to that described for other types of protein inhibitors of serine proteinases.