Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

Thermodynamic characterization of an intermediate state of human growth hormone.

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.     

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.     

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.     

Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.     

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.     

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.     

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Spectroscopic and differential scanning calorimetric studies on the unfolding of <Emphasis Type="Italic">Trichosanthes dioica</Emphasis> seed lectin. Similar modes of thermal and chemical denaturation

Physico-chemical and unfolding studies have been carried out on Trichosanthes dioica seed lectin (TDSL). The lectin exhibited maximum activity between pH 7.0 and 10.0, which decreased steeply at lower pH. The hemagglutination activity of TDSL was unaffected in the temperature range 4–50°C, but decreased rapidly at higher temperatures. Differential scanning calorimetric studies indicate that thermal unfolding of TDSL is an irreversible process, which could be described by a three-state model. The calorimetric scan recorded at pH 7.0 consists of two transitions, occurring at around 338.6 K, and 342.8 K. In the presence of carbohydrate ligands both these transitions shifted to higher temperatures, suggesting that ligand binding stabilizes the native conformation of the protein. The unfolding temperature was highest at pH 5.0 indicating that TDSL is more stable at acidic pH. Gdn.HCl induced unfolding, monitored by following changes in the intrinsic fluorescence properties of the protein, was also observed to be a three-state process involving an intermediate. CD spectroscopy indicates that the secondary and tertiary structures of TDSL are rather similar at different pH values, indicating that the lectin structure remains essentially unchanged over a wide range of pH.     

Secondary structure formation precedes tertiary structure in the refolding of ribonuclease A.

The kinetics of refolding of ribonuclease A were monitored by circular dichroism (CD), tyrosine fluorescence and absorbance in the -40 to -10 degrees C range using a methanol cryosolvent. The native-like far-ultraviolet CD signal returned in the dead-time of the mixing, whereas the native absorbance and fluorescence signals returned in a multiphasic process at rates several orders of magnitude more slowly. Thus the secondary structure was formed much more rapidly than the tertiary structure. In addition, the absorbance signal showed evidence of an early intermediate in which one, or more, tyrosine residues was in a transiently more polar environment. A total of four kinetic phases were observed by absorbance in refolding, the slowest two of which had energies of activation consistent with proline isomerization. A refolding scheme involving initial hydrophobic collapse, concurrent with secondary structure formation, followed by much slower rearrangement to the native tertiary structure is proposed.     

Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.     

Chemical, thermal and pH-induced equilibrium unfolding studies of Fusarium solani lectin

The effect of urea, guanidine thiocyanate, temperature and pH was studied on the conformational stability of Fusarium solani lectin. Equilibrium unfolding with chemical denaturants showed that the lectin was least stable at pH 12 and maximally stable at pH 8.0 near its pI (8.7). Guanidine thiocyanate (the concentration of denaturant at which the protein is half folded, D1/2 = 0.49 M at pH 12) was found to be an eight times stronger denaturant than urea (D1/2 = 3.88 M at pH 12). The unfolding curves obtained with fluorescence and CD measurements showed good agreement indicating a monophasic nature of unfolding and excluded the possibility of formation of any stable intermediate. The effect of pH on the lectin was found to be unusual as at acidic pH, the lectin showed a flexible tertiary structure with pronounced secondary structure, and retained its hemagglutinating activity. On the other hand, the lectin did not show any loss of conformation or activity upto 70 degrees C for 15 min. Moreover, thermal denaturation did not result in the aggregation or precipitation of the protein even at high temperatures. Thermal denaturation was also carried out in the presence of a low concentration of guanidine thiocyanate. Change in the enthalpy of transition (DeltaHm) varied linearly with transition temperature (Tm), which indicated that the heat capacity (DeltaCp = 3.95 kJ . mol-1 . K-1) of the lectin remained constant during the unfolding.     

Pressure versus temperature unfolding of ribonuclease A: an FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site

FTIR spectroscopy was used to characterize and compare the temperature- and pressure-induced unfolding of ribonuclease A and a set of its variants engineered in a hydrophobic region of the C-terminal part of the molecule postulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two-state, reversible unfolding transition using both pressure and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I' band and the tyrosine band, or the maximum of the band assigned to the beta-sheet structure, corroborate the results of a previous study using fourth-derivative UV absorbance spectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure- and temperature-denaturated states of all the ribonucleases characterized retained some secondary structure. However, their spectral maxima were centered at different wavenumbers, which suggests that pressure- and temperature-denaturated states do not have the same structural characteristics. Nevertheless, there was close correlation between the pressure and temperature midpoint transition values for the whole series of protein variants, which indicated a common tendency of stability toward pressure and heat.     

The effect of methanol and temperature on the kinetics of refolding of ribonuclease A

Unfolded ribonuclease A consists of 20% fast refolding (Uf) and 80% slow refolding material (Us). The latter consists of at least two different forms which refold at different rates. We have used absorbance and fluorescence spectrophotometry to compare the kinetics of refolding in aqueous and aqueous-methanol solutions. At 1 degree C and pH 3.0, the addition of increasing concentrations of methanol (to 50%, v/v) had negligible effect on the rates and amplitudes of the slow refolding Us states. The effect of temperature on the two slow phases of refolding was determined in 35 and 50% methanol. From Arrhenius plots the energies of activation were found to be in the vicinity of 20 kcal/mol for both processes. The results suggest that both slow phases correspond to proline isomerization, and that the presence of methanol does not significantly perturb the overall refolding process. It is possible that the faster of the slow refolding phases corresponds to the isomerization of a proline residue which is trans in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.