Intrinsic viscosity of ovomucoid in random coil conformation.

Ovomucoid is denatured by concentrated solutions of guanidine hydrochloride. The intrinsic viscosities of the glycoprotein in 6 M guanidine hydrochloride in the absence and presence of beta-mercaptoethanol were found to be 8.1 and 16.0 ml/g, respectively. Ovomucoid with disulphide bonds reduced exists in linear random coil conformation. However, the intrinsic viscosity of the randomly coiled protein was less than that predicted from the empirical equations describing the molecular weight dependence of intrinsic viscosities of random coil proteins in 6 M guanidine hydrochloride. On excluding the carbohydrate content of the protein, which is theoretically justified, the calculated intrinsic viscosity interestingly became closer to the measured one. The temperature dependence of the intrinsic viscosity of ovomucoid in linear random coil conformation was studied in the temperature range, 25-55 degrees. The features of the intrinsic viscosity-temperature profile are not comparable with those exhibited by other linear random coil proteins in 6 M guanidine hydrochloride.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

A guanidine hydrochloride induced change in ribonuclease without gross unfolding

Although denaturation of ribonuclease by guanidine hydrochloride to a random coil has been considered to be a simple two-state mechanism, the time dependence of our calorimetric data indicate that a cooperative endothermic pretransition may occur near 1.25 M. guanidine hydrochloride (pH 6 and 25°C) without gross unfolding of the protein. Reexamination of other observables as a function of guanidine hydrochloride concentrations as well as activity measurements suggests the possibility of some process other than simple binding occurring in the concentration range below the onset of gross denaturation.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.     

The denaturation of covalently inhibited swine pepsin.

Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

Nuclear magnetic resonance studies of the denaturation of ubiquitin.

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.     

IR spectra of cytochrome c denatured with deuterated guanidine hydrochloride show increase in beta sheet

Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra are obtained for horse heart ferricytochrome c in solutions of 0-7M guanidine hydrochloride and deuterated guanidine hydrochloride. Substitutions of deuterium for hydrogen in both the denaturant and protein provide resolvable amide I spectra over a wide range of denaturant concentrations. Deuteration enhances the ability to measure the true protein IR spectrum in the amide I region in which the secondary structure can be deduced, because spectra in D(2)O are less prone to spectral distortion upon background denaturant subtraction than spectra in H(2)O. Other investigators studying equilibrium unfolded cytochrome c were limited to guanidine concentrations below 3.0M because of detector saturation. Detector saturation is avoided with the use of ATR-FTIR spectroscopy, allowing one to obtain protein spectra at high denaturant concentrations. Second derivative spectra of samples show reductions in alpha helix and increases in beta sheet at high denaturant concentrations, contrary to expectations of finding primarily a random coil secondary structure. Using this new technique, the protein was estimated to consist of 51% beta sheet and only 15% random coil in the presence of 6.6M deuterated guanidine hydrochloride.     

Molecular weight of Escherichia coli β-galactosidase in concentrated solutions of guanidine hydrochloride

The molecular weight of Escherichia coli beta-galactosidase was determined in 6m- and 8m-guanidine hydrochloride by meniscus-depletion sedimentation equilibrium, sedimentation velocity and viscosity. Sedimentation equilibrium revealed heterogeneity with the smallest component having a molecular weight of about 50000. At lower speeds, the apparent weight-average molecular weight is about 80000. By use of a calculation based on an empirical correlation for proteins that are random coils in 6m-guanidine hydrochloride, sedimentation velocity gave a molecular weight of 91000, and the intrinsic viscosity indicated a viscosity-average molecular weight of 84000. Heating in 6m-guanidine hydrochloride lowered the viscosity of beta-galactosidase in a variable manner.     

Molecular weight and hydrodynamic properties of apolipoprotein B in guanidine hydrochloride and sodium dodecyl sulfate solutions.

The apolipoprotein B polypeptide of human serum low density lipoprotein exists (after reduction of disulfide bonds) as a random coil with a molecular weight of 250,000 in concentrated solutions of guanidine hydrochloride. With intact disulfide bonds, there is a limited restraint on the polypeptide conformation in this denaturing solvent. In the presence of saturating amounts of bound sodium dodecyl sulfate, the apolipoprotein is dimeric and highly asymmetric. This work substantiates the monomeric molecular weight of 250,000 for apolipoprotein B reported by others (Smith, R., Dawson, J.R., and Tanford, C. (1972) J. Biol. Chem. 247, 3376-3381) and demonstrates that the dimeric state of the polypeptide extant in vivo is maintained in micellar detergent solution.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride.

The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.     

Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.     

A method for the measurement of the sedimentation coefficient and molecular weight of microgram quantities of proteins in 6 M guanidine hydrochloride

A technique has been perfected for measuring the sedimentation coefficient of microgram quantities of a reduced protein in 6 M guanidine hydrochloride. The protein is sedimented through a gradient of 5-8 M guanidine-HCl in the presence of dithiothreitol in a SW 50.1 swinging-bucket rotor. Run conditions are calibrated by a simultaneous measurement using a single reference protein. Thus, the need for running a calibration curve involving several standard proteins simultaneously with a sample is eliminated. Because of the trace quantity of protein used, the technique yields an estimate of the sedimentation coefficient at zero concentration (s0) directly without extrapolation. Since s0 is a function of the molecular weight of a reduced protein in this solvent, the method also allows an estimate of the subunit molecular weight of the protein. The results of the application of the method to known proteins are reported.     

Integrity of polypeptide chains of spectrin from human erythrocytes.

The suggestion that the high molecular weight erythrocyte membrane protein, spectrin, consists of subunits resistant to dissociation by both sodium dodecyl sulfate and 6 m guanidine hydrochloride has been reevaluated. By gel electrophoresis in dodecyl sulfate and thin-layer gel filtration in 6 m guanidine hydrochloride as well as in the much more powerful denaturant guanidine thiocyanate, and by sedimentation velocity in 6 m guanidine hydrochloride, the molecular weight emerges in the range 2–2.5 × 10⁵. Denaturation profiles as a function of guanidine hydrochloride concentration, observed by circular dichroism, reveal that the spectrin conformation is unusually labile, with a mid-point for the unfolding process at a denaturant concentration near 1 m. Complete acylation with succinic anhydride, as well as reaction with citraconic anhydride, leaves the molecular weight unchanged even in 6 m guanidine hydrochloride. The possibility of measuring molecular weights of proteins by viscosity determination in trifluoroacetic acid was explored. A calibration with a series of proteins gave a Mark-Houwink plot with high scatter, which did not result from low precision of viscosity determination or protein degradation. Evidence is adduced from infrared spectra that the scatter is due to a variable degree of protonation of the polypeptide backbone in the acid, leading to altered hydrodynamic characteristics. Within the semiquantitive limits of the method, spectrin is not further disaggregated in trifluoroacetic acid. The presence of refractory noncovalent interactions and of covalent cross-links has been variously invoked to explain an apparent microheterogeneity in spectrin preparations. The results here described appear to render the former explanation untenable.     

NATIVE AND REGENERATED BOVINE ALBUMIN : I. PREPARATION AND PHYSICOCHEMICAL PROPERTIES

1. Whole bovine albumin, homogeneous in diffusion and sedimentation, and essentially homogeneous in electrophoresis, has been prepared by a method involving ammonium sulfate precipitation of the globulins in the cold and of the albumin at room temperature, isoelectric precipitation of the euglobulins, and reprecipitation of the albumin. 2. The product has been characterized by chemical analysis and by viscosity, diffusion, sedimentation, and electrophoresis measurements. The carbohydrate content is 0.38 per cent, the nitrogen content, 15.2 per cent. The molecular shape approximates that of a prolate ellipsoid with an axial ratio of 3.1, assuming 33 per cent hydration; the average molecular weight is 65,000. 3. Bovine albumin is readily denatured by concentrated solutions of urea or guanidine hydrochloride, gross changes in molecular shape resulting. 4. Regeneration of bovine albumin denatured in solutions of 8 M urea or guanidine hydrochloride yields a material closely resembling the native in carbohydrate content, in molecular size and shape, and in electrophoretic properties. However, the regenerated protein differs from the native in susceptibility to tryptic digestion, and, in this respect, appears to be in a denatured state. 5. In 8 M solutions of guanidine hydrochloride a limiting yield of regenerated albumin equivalent to 95 per cent of the original protein is approached. 6. Bovine crystalbumin, a crystalline carbohydrate-free fraction of the whole albumin, appears to be more susceptible to denaturation than whole bovine albumin.     

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

The structural characteristics of the extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 (PS2) core antenna complexes CP43 and CP47 were investigated using fluorescence emission and circular dichroism (CD) spectra. The extra-membrane domains of CP43 and CP47 possessed a certain degree of secondary and tertiary structure and not a complete random coil conformation. The tertiary structure and the chlorophyll (Chl) a microenvironment of CP47 were more sensitive to guanidine hydrochloride (GuHCl) than that of CP43. Changes in energy transfer from β-carotene to Chl a corresponded well to changes in the tertiary structure while their correlation with changes in the secondary structure was rather poor. Unlike most of water-soluble proteins, both CP43 and CP47 are partly resistant to denaturation induced by guanidine hydrochloride (GuHCl); the denaturation of CP43 or CP47 is not a two-state process. Those features most probably reflect their character as intrinsic membrane proteins.     

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.     

Urea and guanidine hydrochloride induced dissociation and denaturation of bacteriophage F2 capsid.

A single, low molecular weight protein is found after urea or guanidine hydrochloride (Gdn.HCl) treatment of empty capsids derived from bacteriophage f2. The final product of denaturation is apparently a monomer, existing as a random coil in larger than or equal to 4.0 M Gdn.HCl but in a less extended form in 8.0 M urea. In contrast, an 11 S protein component is isolated after treatment of the intact virus with 4.0 M Gdn.HCl (Zelazo & Haschemeyer, 1969), indicating that RNA plays a role in stabilizing larger subunits. Denaturation by Gdn.HCl occurs in two stages as measured by changes in CD and Stokes radius: dissociation that involves a structural perturbation of aromatic side chains, followed by a major, cooperative transition that evidently results in the loss of all noncovalent structure. Denaturation by urea appears to be a much less cooperative process that occurs in several steps over a wide range of urea concentration (1--7 M). In both urea and Gdn.HCl, dissociation into subunits begins at a lower concentration of denaturant than the major changes in conformation.