Alcohol-induced conformational transitions in ervatamin C. An alpha-helix to beta-sheet switchover

Alcohol-induced conformational transitions of erv C, a highly stable cysteine protease, were followed by CD, fluorescence, and activity. At acidic pH, the addition of different alcohols caused two types of conformational transitions. Increasing the concentration of nonfluorinated alkyl alcohols induced a conformational switch from -helix to -sheet. Under these conditions, the protein lost its proteolytic activity and tertiary structure. The switch was a sudden one, observed in 50% methanol, 45% ethanol, and 40% propanol. Under similar conditions of pH and concentration, however, glycerol and TFE enhanced the -helicity of the protein. Methanol-induced denaturation was observed to occur in two stages; the first is the -sheet state stabilized at low alcohol concentrations, and the other is the -sheet state with enhanced ellipticity stabilized at high alcohol concentrations. This -sheet conformation can be attained from the native as well as 6 M GuHCl-denatured state by addition of methanol and exhibits properties different from the native or unfolded state. This state shows loss of tertiary structure and activity, enhanced nonnative secondary structure, noncooperative temperature unfolding, and higher stability toward denaturants as compared to the native state, which are characteristic of the molten globule-like state or O-state, and thus this state may be functioning as an intermediate in the folding pathway of erv C.     

Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.     

Unfolding of ervatamin C in the presence of organic solvents: sequential transitions of the protein in the O-state

The folding of ervatamin C was investigated in the presence of various fluorinated and non-fluorinated organic solvents. The differences in the unfolding of the protein in the presence of various organic solvents and the stabilities of O-states were interpreted. At pH 2.0, non-fluorinated alkyl alcohols induced a switch from the native alpha-helix to a beta-sheet, contrary to the beta-sheet to alpha-helix conversion observed for many proteins. The magnitude of ellipticity at 215 nm, used as a measure of beta-content, was found to be dependent on the concentration of the alcohol. Under similar conditions of pH, fluorinated alcohol enhanced the intrinsic a-helicity of the protein molecule, whereas the addition of acetonitrile reduced the helical content. Ervatamin C exhibited high stability towards GuHCl induced unfolding in different O-states. Whereas the thermal unfolding of O-states was non-cooperative, contrary to the cooperativity seen in the absence of the organic solvents under similar conditions. Moreover, the differential scanning calorimetry endotherms of the protein acquired at pH 2.0 were deconvoluted into two distinct peaks, suggesting two cooperative transitions. With increase in pH, the shape of the thermogram changed markedly to exhibit a major and a minor transition. The appearance of two distinct peaks in the DSC together with the non-cooperative thermal transition of the protein in O-states indicates that the molecular structure of ervatamin C consists of two domains with different stabilities.     

Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study

Alpha-actinin is a cytoskeleton-binding protein involved in the assembly and regulation of the actin filaments. In this work molecular dynamics method was applied to investigate the mechanical behaviour of the human skeletal muscle α-actinin. Five configurations were unfolded at an elongation speed of 0.1 nm/ps in order to investigate the conformational changes occurring during the extension process. Moreover, a sensitivity analysis at different velocities was performed for one of the R2–R3 spectrin-like repeat configuration extracted in order to evaluate the effect of the pulling speed on the mechanical behaviour of the molecule. Two different behaviours were recognized with respect to the pulling speed. In particular, at speed higher than 0.025 nm/ps a continuous rearrangement without evident force peaks was obtained, on the contrary at lower speed evident peaks in the range 500–750 pN were detected. R3 repeat resulted more stable than R2 during mechanical unfolding, due to the lower hydrophobic surface available to the solvent. The characterization of the R2–R3 units can be useful for the development of cytoskeleton network models based on stiffness values obtained by analyses performed at the molecular level.     

Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy

Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium‐binding protein exhibits a broad range of structural transitions, including domain opening and folding–unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well‐known, it has been challenging to examine the dynamics of transition between these structures in real‐time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C‐terminal domain in response to Ca²⁺ binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation‐sensitive tyrosine residues, opening a path to time‐resolved studies of CaM dynamics using Raman spectroscopy.     

Unfolding pathways of human serum albumin: evidence for sequential unfolding and folding of its three domains

Human serum albumin (HSA) contains three alpha-helical domains (I-III). The unfolding process of these domains was monitored using covalently bound fluorescence probes; domain I was monitored by N-(1-pyrene)maleimide (PM) conjugated with cys-34, domain II was monitored by the lone tryptophan residue and domain III was followed by p-nitrophenyl anthranilate (NPA) conjugated with Tyrosine-411 (Tyr-411). Using domain-specific probes, we found that guanidium hydrochloride-induced unfolding of HSA occurred sequentially. The unfolding of domain II preceded that of domain I and the unfolding of domain III followed that of domain I. In addition, the domains I and III refolded within the dead time of the fluorescence recovery experiment while the refolding of domain II occurred slowly. The results suggest that individual domain of a multi-domain protein can fold and unfold sequentially.     

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.     

Allosteric serine hydroxymethyltransferase from monkey liver: Temperature induced conformational transitions

The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°–70°(T _m= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot ofh values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with anh value of 1.7 in the temperature range of 45°–60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.     

Secondary structure of prothymosin alpha evidenced for conformational transitions induced by changes in temperature and concentration of n-dodecyltrimethylammonium bromide

Conformational changes of prothymosin alpha (ProTalpha) induced by changes in temperature and concentration of the denaturant n-dodecyltrimethylammonium bromide (C12TAB) were studied by difference spectroscopy. The conformational transition of ProTalpha by C12TAB was followed as a function of denaturant concentration by absorbance measurements at 230 nm and the data were analyzed to obtain the Gibbs energy of the transition in water (deltaG0(w)) and in a hydrophobic environment (deltaG0(hc)) for saturated protein-surfactant complexes. The value of deltaG0(w) was 6.38 kJ mol(-1) and that for deltaG0(hc), which is not affected by temperature, was -18.62 kJ mol(-1). Changes of absorbance at 230 nm of ProTalpha with temperature can be assumed to resemble a transition in the secondary structure. The parameters characterizing the thermodynamics of unfolding, melting temperature (Tm), enthalpy (deltaHm), entropy (deltaSm) and heat capacity (deltaCp) were determined. The values obtained for Tm, deltaHm, and deltaSm are smaller that those found for other globular proteins; deltaCp was found to be much smaller. These results suggest that ProTalpha exhibits some type of secondary structure under these conditions (10 mM glycine buffer, pH 2.4).     

Simple sequential model for the kinetics of conformational transitions of oligomeric helices and proteins

A simple sequential model is developed which is applicable to the kinetics of melting of some types of oligomeric helices and as an idealization to the kinetics of unfolding of some protein molecules. A procedure is presented for calculating the concentrations of all conformational species as functions of time. The time course of experimentally observable quantities which depend on these concentrations may then be computed. One of the most characteristic features of the model is the distinction between a transient and a steady-state phase. During the latter all molecular parameters change at the same rate, which depends strongly on the difficulty of nucleation and the length of the sequence. Simple approximations to the steady-state rate are discussed in terms of the exact solution. Rates of transient processes dependless strongly on the rate of nucleation and the number of steps and are a more direct reflection of the rates of the rates of the elementary process of propagation. The value of experimental observation of transient process is emphasized.     

Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains

Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits [Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., & Eisenberg, D. (1986) Nature (London) 323, 304-309]. Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl. No dissociation or aggregation of dodecamer occurred at high temperatures. The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis). Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of [Mn2+] studied and with average delta HvH values of 80 and 94 kcal/mol, respectively. These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model). However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating. By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free [Mn2+] from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free [Mn2+] from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme. The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS. Adding substrates [ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP] to Mn.GS increased Tm to varying extents by preferential binding to the folded form. Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C. Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of the hydrophobic core of ribonuclease A through sequential coordinated conformational transitions

With steady-state and time-resolved fluorescence energy-transfer measurements, we determined the distributions of intramolecular distances in nine mutants to study the conformations of wild-type ribonuclease A in the reduced state under folding conditions. Although far-UV-CD measurements show no evidence for a secondary-structure transition, temperature- and GdnHCl-induced changes in intramolecular distance distributions in the reduced state revealed evidence for long-range subdomain structures in the denatured protein. These poorly defined structures, reflected here by wide distributions corresponding to a wide range of energies, form during refolding in a complex sequence of multiple subdomain transitions. A more well-defined structure emerges only when this structural framework, which directs the successive steps in the folding process, matures and is reinforced by stronger interactions such as disulfide bonds.     

Characterization of the protein unfolding processes induced by urea and temperature

Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the β-sheets, while leaving the α-helices almost intact; on the contrary, high temperature preserved the β-sheets and led to a complete loss of the α-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replica-exchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent.     

Comparative study of the conformational transitions of frog and rabbit phosphorylases B

1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectometry. 2. The experimental results of the 1H-2H exchange were expressed in terms of the probability P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy delta Go. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.     

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.     

Glycation induced conformational transitions in cystatin proceed to form biotoxic aggregates: A multidimensional analysis

Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant β structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.     

Molecular dynamics simulation of temperature induced unfolding of animal prion protein

To elucidate the structural stability and the unfolding dynamics of the animal prion protein, the temperature induced structural evolution of turtle prion protein (tPrPc) and bank vole prion protein (bvPrPc) have been performed with molecular dynamics (MD) simulation. The unfolding behaviors of secondary structures showed that the α-helix was more stable than β-sheet. Extension and disruption of β-sheet commonly appeared in the temperature induced unfolding process. The conversion of α-helix to π-helix occurred more readily at the elevating temperature. Furthermore, it was suggested in this work that the unfolding of prion protein could be regulated by the temperature.

Quasi-elastic light scattering study of salt induced conformational transitions of chromatin subunit

The translational diffusion coefficient D_T of monodisperse solutions of 146 base pairs (bp) core particles was studied by the quasi-elastic light scattering technique. When the salinity was raised a change of D_T from 1.9 × 10^?7 cm² s^?1 to 3.2 × 10^?7 cm² s^?1 was detected at about 2 mM NaCl, followed by a smooth decrease of D_T beyond 0.6 M NaCl. The measurements of particle concentration and scattering vector effects on the D_T showed that the influence of interactions between particles can be disregarded. The interaction between particles and counterions is also discussed and does not appear to be the origin of the actual changes in D_T. These transitions of D_T are hence related to changes of shape and size of the particles. It is shown that the single transition at low salinity corresponds to a conformational change while the variation of D_T at high salinity can be interpreted by a destabilization of the edifice. In different regions of salinities, the observed values of D_T can lead to reasonable hydrodynamic models.