A study of interactions of carbonic anhydrase B with water and urea. I. Spin-diffusion parameters that determine individual and cooperative properties of molecules

The rigidity parameter (G), which is characteristic of protein compactness, was studied in native globular carbonic anhydrase B. The dependence of parameter G on power and excitation time of spin-diffusion was expressed analytically. We found out that native carbonic anhydrase B is able to form water-protein units that are probabilistically distributed with respect to their sizes. Large water-protein units can be detected by analyzing the spin-diffusion spectra. The excitation frequencies of spin-diffusion spectra were shifted far away from typical 1H NMR spectra of carbonic anhydrase B.     

A study of interaction of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 M urea

Formation of the associates of carbonic anhydrase B (pH 5.7, 4.2 M urea, and T = 297 K) as a function of protein concentration and time clapsed after preparation of solutions was studied by nuclear magnetic resonance spectroscopy (spin diffusion method). It was demonstrated that the association was a two-stage process. The initial (fast) stage, involving the formation of persistent blocks, was independent of the time elapsed after the solution preparation. A urea concentration of 4.2 M allows the protein molecules to interact with one another to form rather small persistent blocks in combination with solvent molecules, so that the mobility of each molecule remains nearly unchanged. The final (slow) stage is time-dependent and involves the formation of large structures from the persistent blocks. It is shown that parameters G* and S*, which characterize spin diffusion (in protein and solvent, respectively) when it is excited at frequencies remote from the NMR spectral signals, are related to the size probability distribution of the solvent-protein associates and are determined by their collective properties.     

Studies of interactions of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 M of urea

The formation of carbonic anhydrase B associates (pH 5.7, urea concentration 4.2 M, 297 K) was studied as a function of protein concentration and time by nuclear magnetic resonance spectroscopy (spin diffusion method). It was found that the association process proceeds in two steps. The first step is relatively fast and cannot be controlled by our methods. During this step, persistent units are built. These consist of protein molecules that are able to interact with solvent molecules and with each other when protein solution contains 4.2 M of urea. Persistent units are relatively small (two, three protein molecules), and their mobility matches one of a single protein. The second step is slower, and throughout this step large structures are formed from persistent units. The parameters G* and S*, which characterize spin diffusion in a protein and a solvent, respectively (when spin diffusion excitation happens away from NMR spectral signals) are related to the probable size distribution of protein-solvent associates and are determined by their collective properties.     

Dynamics of oligomer formation by denatured carbonic anhydrase II

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.     

Carbonic Anhydrase B Interactions with Water and Urea

High-resolution NMR spectroscopy has been used to study native carbonic anhydrase B unfolding with urea at pH 5.75 and T = 298 K. The rigidity parameter reflecting the effectiveness of spin diffusion (SD) displays a sigma-like dependence on urea concentration, which is characteristic of denaturing processes. The ratio between the integral intensities of urea and protein signals measured in SD spectra and normal 1D spectra are the same. This suggests the absence of a predominant interaction between urea and protein molecules. The concentration of large protein-solvent complexes rapidly increases at urea concentrations of 4.2–6.2 M, which is apparently related to the transition of the protein into the molten globule state. If the urea concentration is increased to 6.6 M, these complexes dissociate, and the polypeptide chain of carbonic anhydrase B becomes completely unfolded.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 497–503.Original Russian Text Copyright © 2005 by Prokhorov, Kutyshenko, Khristoforov.     

Water-protein interactions in the molten-globule state of carbonic anhydrase b: an NMR spin-diffusion study

We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on-resonance signals either at the methyl-proton or at the water-proton signals. The spin-diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein-water interaction in the molten-globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten-globule and denatured states, it remains almost as compact in its molten-globule state as it is in the native state. The spin-diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water.     

Protein stabilization by urea and guanidine hydrochloride

The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.     

Urea denaturation of barnase: pH dependence and characterization of the unfolded state.

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken.     

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.     

THE TEMPERATURE COEFFICIENT OF THE UREA DENATURATION OF EGG ALBUMIN

Evidence is brought forward to show that at concentrations of urea high enough to split the egg albumin molecule the solubility changes produced by urea are profoundly modified. The degree of precipitation after dialysis is the net result of two changes produced by the urea: the first, normally spoken of as denaturation, which makes the protein insoluble in dilute solution and the second, a splitting of the molecule which makes it soluble. These two reactions may proceed independently and simultaneously or the second reaction may follow the first, taking place in the denatured molecule only. In view of the decrease in the opalescence with time, the latter process is more probable. Both of these reactions have positive temperature coefficients, but as the concentration of urea increases the second reaction is more affected by increase in temperature than the first, and consequently the resulting opalescence decreases rather than increases with temperature. This accounts for and explains reports of negative temperature coefficients of denaturation, when denaturation is measured by the amount of insoluble material found on dilution. The occurrence of these two reactions, one leading to an increase and the other to a decrease in the amount of insoluble protein, should be taken into account when denaturation changes in egg albumin with urea are studied.     

Denaturation of concanavalin A by urea at acid pH

The denaturation of dimeric concanavalin A induced by urea at pH 3 has been studied using optical activity and sedimentation velocity. Under the conditions employed Mn+2 and Ca+2 are dissociated from the protein, but the basic structural elements are little changed from those prevailing in the functional lectin at pH 5.5 [H.E. Auer and T. Schilz, preceding paper in this issue]. The protein passes through three stages as the urea concentration is varied from 0 to 10 M. Below 4 M urea the only effect observed is the loss of optical activity of the aromatic amino acid residues. At 4 M, a conformational change occurs producing extensive aggregation, which persists to 7 M. At 8-10 M urea a disordered monomeric protein molecule prevails. The protein could be reactivated provided that dilution to native conditions was very rapid and the protein concentration remained very low. Kinetics of denaturation were monitored by optical activity at 218, 225 and 283 nm. Transients with one, two or three components were observed, which were resolved by nonlinear regression according to sequential first-order decay laws. First order character was confirmed by independence of the kinetic parameters from protein concentration over a two- to four-fold range. Enthalpies and entropies of activation for the various steps were also determined. The transients at the three wavelengths monitor changes in beta structure, beta turns and aromatic groups, respectively. The urea dependence of the rate constants is unique in most cases. It is concluded that different structural elements of the concanavalin A molecule unfold independently from one another.     

Impact of denaturation with urea on recombinant apolipoprotein A-IMilano ion-exchange adsorption: equilibrium uptake behavior and protein mass transfer kinetics

We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I(Milano) (apo A-I(M)) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic alpha helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I(M) mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 x 10(-9) cm2/s in the absence of urea to 6.0 x 10(-8) cm2/s when apo A-I(M) is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins—carbonic anhydrase B—is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.     

Determination of the concentration of protein by dry weight--a comparison with spectrophotometric methods

A protocol for dry weight determination of the concentration of protein, using 0.2-1.0 mg of protein per sample, has been presented and applied to nine proteins: bovine serum albumin, ovalbumin, bovine carbonic anhydrase B, galactoside binding protein (rabbit), lens calinaris lectin B, green pea lectin, soy bean agglutinin-m, wheat germ agglutinin, and antithrombin III. Dry weights, combined with spectrophotometry, have been used to calculate A1% 1 cm values at 280 nm for these proteins in dilute salt solutions and are compared with published values. From absorptivities at 288 and 280 nm in 6 M guanidine hydrochloride, the number of tryptophan residues per molecule has also been calculated and compared with literature values. These results demonstrate the utility of the present method of dry weight determination.     

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.     

Regulation of the rate of urea synthesis in liver by extracellular pH. A major factor in pH homeostasis in mammals

The views that catabolism of protein leads to net production of acid and that urinary excretion of ammonium ion represents an equimolar excretion of proton are not compatible with basic chemical relationships (Atkinson, D.E., and Camien, M.N. (1982) Curr. Top. Cell. Regul. 21, 261-302). Metabolism of protein produces significant amounts of base (bicarbonate), which is disposed of in the synthesis of urea. In perfused rat liver and in isolated rat hepatocytes, the rate of urea synthesis increases with increase in pH but is not affected by change in the concentration of bicarbonate when pH is held constant. An increase in the concentration of ammonium ion in the suspending medium causes an increase in the rate of urea synthesis by hepatocytes when lactate is the energy source, as previously reported by others, but causes a decrease in the rate of urea synthesis during incubation with glucose or with no added energy source. The rate of urea synthesis decreases when glucose is added to lactate medium. All of these observations are consistent with the view that disposal of bicarbonate is a major function of urea synthesis, and that regulation of the rate of ureagenesis is an important factor in the maintenance of pH homeostasis.     

Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.     

An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions.

Previous work has shown that at neutral pH ferricytochrome c (horse heart) retains certain residual structures in concentrated solutions of urea or guanidine hydrochloride (Tsong, T. Y. (1974), J. Biol. Chem. 249, 1988). Present studies reveal that cooperative unfolding of these residual structures can be achieved by acidification of the protein to pH 4 in 9 M urea but can only be partially achieved in a 6 M guanidine hydrochloride solution. The evidence that the residual structures unfold in 9 M urea upon acidification is twofold. (1) Further uncoupling of the Trp-59-heme interaction occurs; this is reflected in the intensification of the tryptophan fluorescence from 55 to 90 percent relative to that of free tryptophan in the same solvent. (2) The intrinsic viscosity of the protein solution increases from 15.0 to 21 ml/g. The acidification also induces a spin-state transformation of the heme group at pH 5 both in urea and in guanidine hydrochloride. Acidic titration of the protein in urea and guanidine hydrochloride indicates that the unfolding involves the absorption of a single proton. However, the kinetics of the spin-state transformation are triphasic. These results suggest that the displacement of the ligand His-18 by a solvent molecule and the subsequent disintegration of the residual structures are complex processes and involve at least three kinetic steps. The ineffectiveness of guanidine hydrochloride as a denaturant for ferricytochrome c is shown to be due to the presence of the high concentration of Cl minus which can stabilize certain elements of the protein structure.     

Kidney retention of urea in sheep on a hypoprotein diet: study by retrograde perfusion of urea in the kidney pelvis

In order to study the role of the renal pelvis on urea sparing in sheep fed low protein diets, the pelvis was perfused through the ureter with 1M and 3M urea solutions. Eight ewes were used: four on a regular diet (total nitrogen 188.7 g.kg-1 dry matter) and the other four on a low protein diet (total nitrogen 109.4 g.kg-1 dry matter). On each animal, perfusions were performed on one kidney; the other one was kept as a control. Fractional excretion of urea (TEu) and urea (Cu), inulin, para-aminohippurate and osmolar clearances, were determined during five experimental periods of 30 min each (T = control, 1M = perfusion with 1M urea solution, R1 = first period of recovery, 3M = perfusion with 3M urea solution, R2 = second period of recovery). 1. During control periods sheep on low protein diet have a greater capacity of urea retention than sheep on regular diet, under antidiuretic conditions (inulin U/P = 200). The following data (means +/- S.D.) are all reduced in animals on low protein diet: TEu by 36% (0.38 +/- 0.19 vs. 0.59 +/- 0.28 for normal protein sheep, p less than 0.05), Cu by 55% (0.50 +/- 0.19 vs. 1.15 +/- 0.49 ml.min-1.kg-1 for normal sheep, p less than 0.01) and amount of urea excreted by 80% (2.1 +/- 0.7 vs. 10.4 +/- 2.7 mg.min-1 for normal sheep, p less than 0.01). 2. The linear regression analysis of the relationship between tubular reabsorption of urea and its filtered amount shows that the capacity of urea retention is significantly higher in low protein sheep and that the difference between the two groups is greater as the filtered amount increases. Following 1M and 3M perfusions, the capacity of urea reabsorption by the perfused kidneys is significantly decreased in low protein animals whereas there is no change in the normal ones. The result is that perfused kidneys of the low protein sheep increase the amount of urea excreted during these periods: urine concentration of urea (Uu) increases by 55% during R1 and by 144% during R2, TEu increases by 60% during R1 and by 147% during R2 and Cu increases by 40% during R1 and by 95% during R2, without any variation of urine flow rate. These changes could be understood, provided that an important transfer of the perfused urea to the renal medulla in the low protein sheep would reduce the concentration gradients which enhance urea passive reabsorption from the collecting ducts.(ABSTRACT TRUNCATED AT 250 WORDS)