Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Structural characterization of protein-denaturant interactions: crystal structures of hen egg-white lysozyme in complex with DMSO and guanidinium chloride

A variety of physico-chemical methods employ chemical denaturants to unfold proteins, and study different biophysical processes involved therein. Chemical denaturants are believed to induce unfolding by stabilizing the unfolded state of proteins over the folded state, either macroscopically or through specific interactions. In order to characterize the nature of specific interactions between proteins and denaturants, we have solved crystal structures of hen egg-white lysozyme complexed with denaturants, and report here dimethyl sulfoxide and guanidinium chloride complexes. The dimethyl sulfoxide molecules and guanidinium ions were seen to bind the protein at specific sites and were involved in characteristic interactions. They share a major binding site between them, the C site in the sugar binding cleft of the enzyme. Although the overall conformations of the complexes were very similar to the native structure, spectacular conformational changes were seen to occur locally. Temperature factors were also seen to drop dramatically in the local regions close to the denaturant binding sites. An interesting observation of the present study was the generation of a sodium ion binding site in hen egg-white lysozyme in the presence of denaturants, which was hitherto unknown in any of the other lysozyme structures solved so far. Loss of some of the crucial side chain-main chain interactions may form the initial events in lysozyme unfolding.     

A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

Coulomb forces control the density of the collapsed unfolded state of barstar

Although it has been recently shown that unfolded polypeptide chains undergo a collapse on transfer from denaturing to native conditions, the forces determining the dynamics and the size of the collapsed form have not yet been understood. Here, we use single-molecule fluorescence resonance energy transfer experiments on the small protein barstar to characterize the unfolded chain in guanidinium chloride (GdmCl) and urea. The unfolded protein collapses on decreasing the concentration of denaturants. Below the critical concentration of 3.5 M denaturant, the collapse in GdmCl leads to a more dense state than in urea. Since it is known that GdmCl suppresses electrostatic interactions, we infer that Coulomb forces are the dominant forces acting in the unfolded barstar under native conditions. This hypothesis is clearly buttressed by the finding of a compaction of the unfolded barstar by addition of KCl at low urea concentrations.     

Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants

Characteristics and properties of the unfolding free energy change, delta G degrees N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (delta epsilon N-U) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of delta epsilon N-U on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of delta G degrees N-U for the three denaturants using nonlinear least-squares fitting of the primary data, delta epsilon versus [denaturant]. The three delta G degrees N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in delta G degrees N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.     

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.     

How do chemical denaturants affect the mechanical folding and unfolding of proteins?

We present the first single-molecule atomic force microscopy study on the effect of chemical denaturants on the mechanical folding/unfolding kinetics of a small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus). Upon increasing the concentration of the chemical denaturant guanidinium chloride (GdmCl), we observed a systematic decrease in the mechanical stability of GB1, indicating the softening effect of the chemical denaturant on the mechanical stability of proteins. This mechanical softening effect originates from the reduced free-energy barrier between the folded state and the unfolding transition state, which decreases linearly as a function of the denaturant concentration. Chemical denaturants, however, do not alter the mechanical unfolding pathway or shift the position of the transition state for mechanical unfolding. We also found that the folding rate constant of GB1 is slowed down by GdmCl in mechanical folding experiments. By combining the mechanical folding/unfolding kinetics of GB1 in GdmCl solution, we developed the “mechanical chevron plot” as a general tool to understand how chemical denaturants influence the mechanical folding/unfolding kinetics and free-energy diagram in a quantitative fashion. This study demonstrates great potential in combining chemical denaturation with single-molecule atomic force microscopy techniques to reveal invaluable information on the energy landscape underlying protein folding/unfolding reactions.     

Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme.

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).     

Studies of the denaturation and partial renaturation of ovalbumin

1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a(0) and b(0) become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1-4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a(0) and b(0) are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride-mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

Effect of protein denaturants on the structure of water: Electron spin resonance spin probe study

The effect of the urea-class of protein denaturants on the structure of liquid water was studied. The method chosen was to monitor the microviscosity of the medium by estimating the reorientational correlation time, τ₈ and proton hyperfine linewidth, WH of an inert stable organic free radical (2, 2, 6, 4-tetramethyl-piperid 4-one-l-oxyl) by measuring its electron spin resonance spectra in aqueous denaturant solutions. Urea, thiourea and guanidinium chloride were found to disrupt water structure efficiently and continuously, with the effect significant at low molarities. Dimethyl urea was found to be somewhat less efficient. The relation between the structure-breaking tendency of the denaturants and their ability to weaken hydrophobic interactions is rationalised.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.     

Preliminary Studies on the Denaturation of Cross-Linked Lysozyme Crystals

When monoclinic lysozyme crystals are fully cross-linked with glutaraldehyde, and then the protein molecules are denatured while in the crystalline state, a single crystal-gel is formed which is a jelly-like crystal of a denatured protein molecule. It is highly disordered, but has crystalline optical and morphological properties and can be renatured to a cross-linked crystal resembling the original crystal as determined from the X-ray diffraction pattern. Experiments with the following denaturants are described: guanidinium chloride, bromoethanol, urea, and lithium chloride.     

Inorganic salt denaturants stabilize ribonuclease against denaturation by urea

The isothermal denaturation of ribonuclease A by mixed denaturant systems was investigated at 25 degrees C. It was observed that low concentrations of lithium chloride stabilize the protein against denaturation by urea, even though the salt itself is a denaturant. This study also provides, for the first time, the most convincing evidence that the lithium chloride denatured ribonuclease A contains some of the native secondary and tertiary structure.     

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.     

Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase: stabilization of an apo-protein by GdmCl

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo-protein was observed. The stabilization of apo-FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea- and guanidinium chloride-induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.     

“Iron priming” guides folding of denatured aporubredoxins

The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys)(4) sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)(4) sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)(4) site must attain the ferric state in order to achieve its native structure.     

Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations

In performing protein-denaturation experiments, it is common to employ different kinds of denaturants interchangeably. We make use of molecular dynamics simulations of Protein L in water, in urea, and in guanidinium chloride (GdmCl) to ascertain if there are any structural differences in the associated unfolding processes. The simulation of proteins in solutions of GdmCl is complicated by the large number of charges involved, making it difficult to set up a realistic force field. Furthermore, at high concentrations of this denaturant, the motion of the solvent slows considerably. The simulations show that the unfolding mechanism depends on the denaturing agent: in urea the β-sheet is destabilized first, whereas in GdmCl, it is the α-helix. Moreover, whereas urea interacts with the protein accumulating in the first solvation shell, GdmCl displays a longer-range electrostatic effect that does not perturb the structure of the solvent close to the protein.     

Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.