Effect of heavy water on protein flexibility

The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.     

Viscosity dependence of the solute quenching of the tryptophanyl fluorescence of proteins

We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal tryptophan residues in cod parvalbumin and ribonuclease T1, as well as the model systems. N-acetyl-L-tryptophanamide and glucagon. For the latter systems, the apparent rate constant, kq(app), for acrylamide quenching shows a typical diffusion-limited behavior. For parvalbumin and ribonuclease T1, however, the viscosity dependence of kq(app) is quite different. There is little change in the kq(app) values on increasing the bulk viscosity from 1 to 10 cP (by addition of glycerol), but a further increase from 10 to 100 cP results in a significant reduction in the kq(app). Both an unfolding mechanism and a quencher penetration mechanism are considered to explain the results. Only the penetration mechanism is found to be consistent, and our data are interpreted as indicating that the rate-limiting step for quenching goes from being that of diffusion through the protein matrix, at low viscosity, to diffusion through the bulk solvent, at high viscosity. By also considering the Kramers' relationship in fitting our data, we are able to obtain insight regarding the coupling between internal fluctuations in the structure of the protein and motion of the bulk solvent. For parvalbumin and ribonuclease T1, the internal dynamics are found to be very weakly coupled to the bulk.     

Nucleotide- and temperature-induced changes in myosin subfragment-1 structure.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.     

Phase transitions in monoglyceride bilayers. A light scattering study.

Thermotropic phase transitions in single planar bilayers of glycerol mono-oleate have been investigated using quasi-elastic light scattering from thermally excited membrane fluctuations. In certain cases both spectroscopic and intensity information were derived from the observations. For solvent-free bilayers transitional changes were observed in several membrane parameters: in tension, viscosity and thickness, in a combination of lipid orientational order parameter and dielectric anisotropy, and in the lateral compression modulus. These changes, particularly those in membrane thickness and in the anisotropy/order combination, were clearly indicative of a chain-melting transition in the lipid molecules. The chain-melting transition temperature was identified as 16.6 +/- 0.03 degrees C (delta T 1/2 = 1.5 degrees C). The other changes tended to cluster around 12.5 and 16.6 degrees C, suggesting that a two-stage transition was involved. Analysis of pretransitional fluctuations in membrane viscosity, based on a Landau approach, suggested that at the transition the membrane was close to a critical point (T = 12.7 degrees C). Less information was accessible for membranes containing n-decane within their structure. In this case, the change in membrane tension was much smaller than in the solvent-free case and the transition was considerably broadened. These effects accord with an increase in 'interactive volume' within the bilayer due to solvent inclusion.     

Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle contraction.     

Phosphorescence evidence for the role of solvent--protein interactions in the energetics of conformational flexibility of liver alcohol dehydrogenase.

When tryptophyl side chains are hidden within relatively inflexible domains of globular proteins, the lifetime of the phosphorescence from these residues provides a measure of the local conformational flexibility. The phosphorescence decay from the tryptophan buried at the base of the nucleotide-binding domain in liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was monitored between 1 and 40 degrees C to determine the energetics associated with the rate of local unfolding. The slow rate at which this process takes place is found to result from a high entropic barrier rather than from the disruption of strong intramolecular interactions. This observation along with the response of the system to solvent perturbations points to the significance of solvent-protein interactions in determining conformational flexibility.     

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin.

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.     

Phosphorescence anisotropy of liver alcohol dehydrogenase in the crystalline state. Apparent glasslike rigidity of the coenzyme-binding domain

Phosphorescence anisotropy from internal tryptophan (Trp) residues in proteins which are in the crystalline state may provide an experimental approach suitable to study the flexibility of rather rigid segments of protein structure. The phosphorescence anisotropy of Trp-314 in liver alcohol dehydrogenase, which is enclosed within the beta-sheet forming the coenzyme-binding domain, was measured with the protein free in solution and in the crystalline state. In contrast to the free protein, where the rotational correlation time reflects the tumbling rate of the whole macromolecule, there is effectively no loss in anisotropy in the crystalline state. At room temperature, the triplet lifetime of 0.5 s implies that the rotational correlation time of the indole side chain must be larger than 1 s. Anisotropy data show that fluctuations of the indole ring about the average position can only be of limited amplitude (cone of semiangle less than 15 degrees) and that the resistance opposed by the beta-sheet to out-of-plane rotational motions is equivalent to a viscosity larger than 2.5 X 10(8) P, a value which confirms the particular rigidity anticipated for such an assembly of secondary structure.     

The Effect of Glycerol on Molecular Mobility in Amorphous Sucrose Detected by Phosphorescence of Erythrosin B

Luminescence from the triplet probe erythrosin B provides spectroscopic characteristics such as emission energy and lifetime that are sensitive to molecular mobility of the local solvent environment. This study investigated how variation in glycerol content influences the properties of an amorphous sucrose matrix by monitoring phosphorescence of erythrosin B over the temperature range from 5 to 100°C. Emission energy, lifetime, and red-edge excitation data revealed that glycerol affected the mobility of amorphous sucrose matrix primarily through plasticization when the mole ratio of glycerol/sucrose was above 0.27, resulting in a decrease in emission energy (ν _p), lifetime (τ) and energy difference (Δν _P) with excitation at the absorption peak and red edge and an increase in the nonradiative, collisional quenching rate k _TS0. At mole ratios ≤0.27 and at temperatures below the matrix T _g, glycerol exhibited an “antiplasticization” effect, as indicated by higher values of emission energy, lifetime, and energy difference and lower values of the matrix collisional quenching rate. Changes in the distribution of emission energy (emission bandwidth) and lifetime, and variation in the emission lifetime vs wavelength showed that glycerol reduced the spectral heterogeneity. These data illustrate the complex effect of a hydrogen bonding solute on the mobility of an amorphous, hydrogen bonded sugar matrix.     

Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.     

Domain structural flexibility in rhodanese examined by quenching of a phosphorescent probe

Rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) is an enzyme composed of two domains with the catalytic site located in the bottom of the crevice formed by the two domains. In this work, rhodanese was labeled at its catalytic site with the phosphorescence probe eosin isothiocyanide. The accessibility of molecules to the probe was determined by phosphorescence lifetime-quenching studies. The phosphorescent probe was much more accessible to small molecules (I- and thiosulfate, radius about 3-5 A) than to a larger molecule (spin-label probe TEMPO, radius about 8-10 A). It was observed that a temperature-induced change in the rate of quenching occurred at around 28 degrees C. The results are interpreted in terms of structural fluctuations and displacement in the domains.     

Excited states of tryptophan in cod parvalbumin. Identification of a short-lived emitting triplet state at room temperature.

The fluorescence and phosphorescence spectra of model indole compounds and of cod parvalbumin III, a protein containing a single tryptophan and no tyrosine, were examined in the time scale ranging from subnanoseconds to milliseconds at 25 degrees C in aqueous buffer. For both Ca- bound and Ca-free parvalbumin and for model indole compounds that contained a proton donor, a phosphorescent species emitting at 450 nm with a lifetime of approximately 20-40 ns could be identified. A longer-lived phosphorescence is also apparent; it has approximately the same absorption and emission spectrum as the short-lived triplet molecule. For Ca parvalbumin, the decay of the long-lived triplet tryptophan is roughly exponential with a lifetime of 4.7 ms at 25 degrees C whereas for N-acetyltryptophanamide in aqueous buffer the decay lifetime was 30 microseconds. In contrast, the lifetime of the long-lived tryptophan species is much shorter in the Ca-free protein compared with Ca parvalbumin, and the decay shows complex nonexponential kinetics over the entire time range from 100 ns to 1 ms. It is concluded that the photochemistry of tryptophan must take into account the existence of two excited triplet species and that there are quenching moieties within the protein matrix that decrease the phosphorescence yield in a dynamic manner for the Ca-depleted parvalbumin. In contrast, for Ca parvalbumin, the tryptophan site is rigid on the time scale of milliseconds.     

Effects of cavity-forming mutations on the internal dynamics of azurin

The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.     

Oxygen and acrylamide quenching of protein phosphorescence: correlation with protein dynamics

Oxygen quenching of protein phosphorescence and activation enthalpies for the structural fluctuations underlying O2 and acrylamide diffusion were determined for RNase T1, glyceraldehyde-3-phosphate dehydrogenase and beta-lactoglobulin, which have the phosphorescing residues located in relatively solvent-exposed and flexible regions of the polypeptide. The results, compared with those obtained for proteins characterised by a very rigid environment, established that kqO2 was directly correlated to the flexibility of the protein matrix surrounding the chromophore. While the migration of acrylamide was characterised by delta H(double dagger), which was strongly dependent on the fluidity of the structure about the Trp residue, the values of the activation enthalpies for the oxygen migration of all the proteins studied were rather similar, approximately 10 kcal mol(-1), in spite of the depth of the chromophore and the rigidity of its environment. The implications of these findings for the migration of small solutes inside proteins have been discussed.     

The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding

We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.     

Substrate-induced conformational changes in the membrane-embedded IIC(mtl)-domain of the mannitol permease from Escherichia coli, EnzymeII(mtl), probed by tryptophan phosphorescence spectroscopy

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII(mtl), of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC(mtl)-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of beta-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII(mtl).     

Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core

The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.     

Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy

Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.     

Diffusional barrier in the unfolding of a small protein

To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.