Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.     

Analysis of hydrodynamic data for denatured globular proteins in terms of the wormlike cylinder model.

Hydrodynamic data, i.e. intrinsic viscosity and sedimentation coefficient, for denatured globular proteins in 6 M guanidinium chloride have been re-analysed in terms of the Yamakawa-Fujii theory of the wormlike cylinder model. Molecular parameters thus obtained ((mean value of R2 0/M) infinity, the Kuhn statistical segment length and the molecular weight per unit contour length) are in better agreement with the values obtained theoretically or by other methods than those evaluated on the basis of the model of non-draining random coil.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Molecular Weight Determination of Sendai and Newcastle Disease Virus RNA

The molecular weights of Sendai and Newcastle disease virus RNA were estimated by sedimentation in sucrose gradients and by length measurements in the electron microscope under both denaturing and nondenaturing conditions. Sedimentation analyses under denaturing conditions yielded molecular weight estimates of 2.3 x 10(6) to 2.6 x 10(6), whereas length measurements yielded estimates of 5.2 x 10(6) to 5.6 x 10(6) for both denatured and nondenatured viral RNA. It would appear that the conditions of denaturation used (99% dimethyl sulfoxide at 26 C, and reaction with 1.1 M formaldehyde for 10 min at 60 C) do not equally denature parainfluenza virus RNA and other RNAs, such as cellular rRNA, 45S rRNA precursor, and R17 RNA.     

Separation of the complementary strands of DNA fragments on polyacrylamide gels.

32P-labeled (in vivo) phiX174 RFI DNA was restricted by Hinc II. Three aliquots of the same digest: a) nondenatured, b) heat denatured, and c) denatured by 5 mM Me-HgOH were analyzed on 3-15% acrylamide gel gradients or on 3% gels with reduced N,N'-methylene-bis-acrylamide. The autoradiography of the gels showed that the nondenatured sample migrates two times faster than the denatured samples. After denaturation each original fragment appeared as a doublet. Using in vitro synthesized RFI DNA labeled only in negative strand with 32P we could identify the position of the negative strand in each denatured doublet. The single strand DNA fragments could be recovered from the gel slices on a semi-preparative scale by electrophoresis into dialysis tubing.     

Protocol to determine accurate absorption coefficients for iron-containing transferrins

An accurate protein concentration is an essential component of most biochemical experiments. The simplest method to determine a protein concentration is by measuring the A₂₈₀ using an absorption coefficient (ε) and applying the Beer-Lambert law. For some metalloproteins (including all transferrin family members), difficulties arise because metal binding contributes to the A₂₈₀ in a nonlinear manner. The Edelhoch method is based on the assumption that the ε of a denatured protein in 6 M guanidine-HCl can be calculated from the number of the tryptophan, tyrosine, and cystine residues. We extend this method to derive ε values for both apo- and iron-bound transferrins. The absorbance of an identical amount of iron-containing protein is measured in (i) 6 M guanidine-HCl (denatured, no iron), (ii) pH 7.4 buffer (nondenatured with iron), and (iii) pH 5.6 (or lower) buffer with a chelator (nondenatured without iron). Because the iron-free apoprotein has an identical A₂₈₀ under nondenaturing conditions, the difference between the reading at pH 7.4 and the lower pH directly reports the contribution of the iron. The method is fast and consumes approximately 1 mg of sample. The ability to determine accurate ε values for transferrin mutants that bind iron with a wide range of affinities has proven to be very useful; furthermore, a similar approach could easily be followed to determine ε values for other metalloproteins in which metal binding contributes to the A₂₈₀.     

NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that approximately 25% of the residues are involved in residual structures. Conformational shifts of the alpha-protons, heteronuclear (15)N-[(1)H] NOEs and (15)N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1-70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone (1)H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.     

Solution and conformational properties of wheat beta-D-glucans studied by light scattering and viscometry

The solution properties of wheat beta-glucan were investigated by light scattering and viscometric methods. The hydrodynamic radius (R(h)), weight average molecular weight (M(w)), radius of gyration (R(g)), and the second virial coefficient (A(2)) of wheat beta-glucan were determined by both dynamic and static light scattering methods, whereas the critical concentrations (c) of the solution were derived from [eta] via viscometric method. The structure sensitive parameters, rho (1.52-1.62), the conformation parameter nu (0.62), and the Mark-Houwink-Sakurada exponents alpha (0.78) confirmed the random coil conformation of wheat beta-glucan in 0.5 M NaOH solution. The characteristic ratio (4.97) was obtained by the random flight model, and the statistical segment length (8.83 nm) was derived from the wormlike cylinder model. It was found that the wormlike cylinder model could explain the chain stiffness better than the random flight model, which suggested an extended random coil conformation of wheat beta-glucan in 0.5 M NaOH solution. The study also revealed that the structure feature of wheat beta-glucan; that is, the higher trisaccharide-to-tetrasaccharide ratio contributed to the stiffer chain conformation compared with other cereal beta-glucans.     

Melting behaviour of a triple helical polysaccharide schizophyllan in aqueous solution

Two sonicated samples of schizophyllan in aqueous solution at temperatures from 20 to 160°C were investigated by viscometry. The temperature dependence of the viscosity coefficient η showed that schizophyllan in water undergoes an irreversible thermal transition at about 135°C. The values of $\text{(ln η}{}_{\mathrm{r}}\text{)}\text{c}$ (ηr is the relative viscosity and c is the polymer concentration (w/v)) at 25°C determined after preheating aqueous schizophyllan indicated that the major conformations of schizophyllan in water at 120 and 150°C are triple helix and single random coil, respectively. Thus, it was concluded that the change in η at about 135°C with an increase in temperature is due to the melting of triple helices to single chains. Schizophyllan denatured to single chains at about 150°C did not restore the intact triple helix, but formed aggregates, when the solution was cooled to 25°C. It was also found that the aggregates form a gel when c is higher than a certain value.     

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.     

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.     

Flory temperature and upper critical solution temperature of gelatin solutions

The Flory temperatures (theta) measured by turbidity experiments performed on gelatin solutions were found to be 12 +/- 0.3, 13 +/- 0.3, 14 +/- 0.3, 14.5 +/- 0.3, and 15 +/- 0.3 degrees C for salt concentrations 0.1, 0.075, 0.05, 0.025, and 0 M (NaCl), respectively. Estimated persistence length (l(p)) of this weakly charged polyelectrolyte could be deduced from the Benoit and Doty (J. Phys. Chem. 1953, 57, 958) relationship with the approximation that this biopolymer assumes a compact near-globular shape at Flory temperature, implying l(p) = 9(R(h))(2)/(5L(m)), where L(m) is the contour length and R(h) is the hydrodynamic radius. It was found that l(p) approximately 2.2 +/- 0.2 nm at room temperature (20 degrees C), invariant of salt concentration. The Flory expansion factor (alpha= R(h)(T)/R(h)(theta) = 1.5+/-0.2) was found to be almost constant. theta-Composition for this biopolymer was deduced from turbidimitric titration of aqueous gelatin solutions with the alcohols methanol, ethanol, 2-propanol, and tert-butyl alcohol. It appears that hydrophobic interactions play a crucial role in causing chain collapse at theta-temperature and composition.     

Fully reduced ribonuclease A does not expand at high denaturant concentration or temperature

The dimensions of a denatured protein, fully reduced ribonuclease A (r-RNase A), have been measured using synchrotron-based small angle X-ray scattering. The radius of gyration, 34-35 A, is unchanged from 0-6 M guanidinium chloride and from 20-90 degrees C at pH 2.5, and agrees with the known scaling behavior for a multitude of chemically denatured states. The polypeptide is behaving as a statistical coil in the non-interacting, high-temperature limit.     

The effect of sodium ion concentration on intrastrand base-pairing in single-stranded DNA.

The salt-induced formation of duplex structure (primarily hairpin loops) in denatured calf thymus DNA was monitored by measuring the decrease in absorbance at 260 nm as a function of increasing sodium ion concentration. It was found that this process was noncooperative and could be accurately described by the mass-action expression for the reversible formation of a binary complex: single strand (coil) + free sodium ion <==> hairpin (with associated sodium ion). The equilibrium constant for the transition was found to be 6 (M Na+)-1. The extrapolated absorbance at infinite salt concentration represents 11% hyperchromicity, which is one third of the hyperchromicity of denatured DNA in the absence of salt (36%).     

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.     

Denatured state thermodynamics: residual structure, chain stiffness and scaling factors

A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum histidine-heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37-83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2+/-0.3. This value is much larger than the scaling factor of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins.     

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 nature of substrate-attached materials in human fibroblast cultures: localization of cell and fetal calf serum components.

Human fibroblasts have been used as an in vitro model to examine the morphology and origin of substrate-attached materials. In cultures of subconfluent cells, no ‘tracks’ or ‘pools’ of material could be detected on substrata by anodic oxide interferometry or electron microscopy. However, a continuous layer of densely staining material was present on Falcon plastic tissue culture dishes never exposed to cells or culture medium. Exposure of substrata to culture medium caused the adsorption of fetal calf serum (FCS) components onto the substratum within a few minutes. Although antigenic FCS components remained on the substrata for several days, they were seldom adsorbed to the cells. The hypothesis was formulated that adhesion was mediated by FCS components on the substrata, but not by cellular materials deposited extracellularly. Support for this hypothesis was obtained by studying serum-dependent differences in cell adhesion. Fibroblasts subcultured in the presence of FCS components were usually separated from the substratum by a distance of at least 30 Å. In the absence of FCS components, the cells were more closely adherent, in the range at which the near van der Walls forces were effective. Fibroblasts subcultured in the absence of serum components could be removed readily from the substratum, leaving lsfootprints’ of cell surface material behind. Although this material has been prepared similarly to ‘microexudates’ from other types of cultured cells, its relationship to those microexudates has not been determined.     

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.     

Dynamic regimes and correlated structural dynamics in native and denatured alpha-lactalbumin

Understanding the mechanisms of protein folding requires knowledge of both the energy landscape and the structural dynamics of a protein. We report a neutron-scattering study of the nanosecond and picosecond dynamics of native and the denatured alpha-lactalbumin. The quasielastic scattering intensity shows that there are alpha-helical structure and tertiary-like side-chain interactions fluctuating on sub-nanosecond time-scales under extremely denaturing conditions and even in the absence of disulfide bonds. Based on the length-scale dependence of the decay rate of the measured correlation functions, the nanosecond dynamics of the native and the variously denatured proteins have three dynamic regimes. When 0.051.0 A(-1) is a regime that displays the local dynamic behavior of individual residues, Gamma proportional to Q(1.8+/-0.3). The picosecond time-scale dynamics shows that the potential barrier to side-chain proton jump motion is reduced in the molten globule and in the denatured proteins when compared to that of the native protein. Our results provide a dynamic view of the native-like topology established in the early stages of protein folding.