The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

Two-dimensional infrared correlation spectroscopy as a probe of sequential events in the thermal unfolding of cytochromes c.

The sequential unfolding events of horse, cow, and tuna ferricytochromes c (cyt c) as a function of increasing temperature over the range 25-81 degrees C were investigated by resolution-enhanced two-dimensional infrared (2D IR) correlation spectroscopy. The 2D IR analysis revealed that in the thermal denaturation of the two mammalian cyts, the overall sequence of unfolding is similar, with denaturation of extended-chain and turn structures occurring prior to unfolding of alpha-helices, followed by denaturation of residual stable extended-chain structures. In tuna cyt c, denaturation of all extended-chain structures precedes the unfolding of alpha-helices. Moreover, in cow cyt c, unfolding of all helical components occurs as one cooperative unit, but in horse and tuna cyts c, the helical components behave as subdomains that unfold separately, as proposed recently by Englander and co-workers for horse cyt c [Bai et al. (1995) Science 269, 192-197; Milne et al. (1999) J. Mol. Biol. 290, 811-822]. At higher temperatures, following the loss of secondary structure, protein aggregation occurs in the three cyts c. The data presented here establish that variations in the thermal unfolding of cyts c can be associated with specific sites in the protein that influence local flexibility yet have little affect on global stability. This study demonstrates the power of resolution-enhanced 2D IR correlation spectroscopy in probing unfolding events in homologous proteins.     

Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein. Role of the 4'-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein

The unfolding pathways of the two forms of Plasmodium falciparum acyl carrier protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation. Both the apo form and the holo form displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provides values for the conformational stability of the two proteins. Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the - DeltaG of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done. The higher stability of the holo form can be attributed to the number of favorable contacts that the 4'-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds. Furthermore, there are several hydrophobic interactions with 4'-phosphopantetheine that firmly maintain the structure of the holo form. We show here for the first time that the interactions between 4'-phosphopantetheine and the polypeptide backbone of acyl carrier protein stabilize the protein. As Plasmodium acyl carrier protein has a similar secondary structure to the other acyl carrier proteins and acyl carrier protein-like domains, the detailed biophysical characterization of Plasmodium acyl carrier protein can serve as a prototype for the analysis of the conformational stability of other acyl carrier proteins.     

An engineered disulfide bond in dihydrofolate reductase

Substitution of cysteine for proline-39 in Escherichia coli dihydrofolate reductase by oligonucleotide-directed mutagenesis positions the new cysteine adjacent to already existing cysteine-85. When the mutant protein is expressed in the E. coli cytosol, the cysteine sulfur atoms are found, by X-ray crystallographic analysis, to be in van der Waals contact but not covalently bonded to one another. In vitro oxidation by dithionitrobenzoate results in formation of a disulfide bond between residues 39 and 85 with a geometry close to that of the commonly observed left-handed spiral. Comparison of 2.0-A-refined crystal structures of the oxidized (cross-linked) and reduced (un-cross-linked) forms of the mutant enzyme shows that the conformation of the enzyme molecule was not appreciably affected by formation of the disulfide bond but that details of the molecule's thermal motion were altered. The disulfide-cross-linked enzyme is at least 1.8 kcal/mol more stable with respect to unfolding, as measured by guanidine hydrochloride denaturation, than either the wild-type or the reduced (un-cross-linked) mutant enzyme. Nevertheless, the cross-linked form is not more resistant to thermal denaturation. Moreover, the appearance of intermediates in the guanidine hydrochloride denaturation profile and urea-gradient polyacrylamide gels indicates that the folding/unfolding pathway of the disulfide-cross-linked enzyme has changed significantly.     

Biphasic reductive unfolding of ribonuclease A is temperature dependent.

The kinetics of the reversible thermal unfolding, irreversible thermal unfolding, and reductive unfolding processes of bovine pancreatic ribonuclease A (RNase A) were investigated in NaCl/Pi solutions. Image parameters including Shannon entropy, Hamming distance, mutual information and correlation coefficient were used in the analysis of the CD and 1D NMR spectra. The irreversible thermal unfolding transition of RNase A was not a cooperative process, pretransitional structure changes occur before the main thermal denaturation. Different dithiothreitol (dithiothreitolred) concentration dependencies were observed between 303 and 313 K during denaturation induced by a small amount of reductive reagent. The protein selectively follows a major unfolding kinetics pathway with the selectivity can be altered by temperature and reductive reagent concentration. Two possible explanations of the selectivity mechanism were discussed.     

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.     

High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.     

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.     

Two-dimensional infrared correlation spectroscopy study of sequential events in the heat-induced unfolding and aggregation process of myoglobin

Unfolding and aggregation are basic problems in protein science with serious biotechnological and medical implications. Probing the sequential events occurring during the unfolding and aggregation process and the relationship between unfolding and aggregation is of particular interest. In this study, two-dimensional infrared (2D IR) correlation spectroscopy was used to study the sequential events and starting temperature dependence of Myoglobin (Mb) thermal transitions. Though a two-state model could be obtained from traditional 1D IR spectra, subtle noncooperative conformational changes were observed at low temperatures. Formation of aggregation was observed at a temperature (50-58 degrees C) that protein was dominated by native structures and accompanied with unfolding of native helical structures when a traditional thermal denaturation condition was used. The time course NMR study of Mb incubated at 55 degrees C for 45 h confirmed that an irreversible aggregation process existed. Aggregation was also observed before fully unfolding of the Mb native structure when a relative high starting temperature was used. These findings demonstrated that 2D IR correlation spectroscopy is a powerful tool to study protein aggregation and the protein aggregation process observed depends on the different environmental conditions used.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.     

Thermal unfolding of Escherichia coli trigger factor studied by ultra-sensitive differential scanning calorimetry

Temperature-induced unfolding of Escherichia coli trigger factor (TF) and its domain truncation mutants, NM and MC, were studied by ultra-sensitive differential scanning calorimetry (UC-DSC). Detailed thermodynamic analysis showed that thermal induced unfolding of TF and MC involves population of dimeric intermediates. In contrast, the thermal unfolding of the NM mutant involves population of only monomeric states. Covalent cross-linking experiments confirmed the presence of dimeric intermediates during thermal unfolding of TF and MC. These data not only suggest that the dimeric form of TF is extremely resistant to thermal unfolding, but also provide further evidence that the C-terminal domain of TF plays a vital role in forming and stabilizing the dimeric structure of the TF molecule. Since TF is the first molecular chaperone that nascent polypeptides encounter in eubacteria, the stable dimeric intermediates of TF populated during thermal denaturation might be important in responding to stress damage to the cell, such as heat shock.     

Three steps in the thermal unfolding of F-actin: an experimental evidence

The development of differential scanning calorimetry has resulted in an increased interest in studies of the unfolding process in proteins with the aim of identifying domains and interactions with ligands or other proteins. Several of these studies were done with actin and showed that the thermal unfolding of F-actin occurs in at least three steps; this was interpreted as the denaturation of independent domains. In the present work, we have followed the thermal unfolding of F-actin using differential scanning calorimetry (DSC), CD spectroscopy, and probe fluorescence. We found that the three steps revealed through DSC are not the denaturation of independent domains. These three steps are a change in the environment of cys 374 at 49.5 degrees C; a modification at the nucleotide-binding site at 55 degrees C; and the unfolding of the peptide chain at 64 degrees C. Previous interpretations of the thermograms of F-actin were thus erroneous. Since DSC is now widely used to study proteins, our experimental approach and conclusions may also be relevant in denaturation studies of proteins in general.     

Biophysical effect of amino acids on the prevention of protein aggregation

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.     

Four-state equilibrium unfolding of an scFv antibody fragment

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.     

Pretransitional structural changes in the thermal denaturation of ribonuclease S and S protein

Two mechanisms have been proposed for the thermal unfolding of ribonuclease S (RNase S). The first is a sequential partial unfolding of the S peptide/S protein complex followed by dissociation, whereas the second is a concerted denaturation/dissociation. The thermal denaturation of ribonuclease S and its fragment, the S protein, were followed with circular dichroism and infrared spectra. These spectra were analyzed by the principal component method of factor analysis. The use of multiple spectral techniques and of factor analysis monitored different aspects of the denaturation simultaneously. The unfolding pathway was compared with that of the parent enzyme ribonuclease A (RNase A), and a model was devised to assess the importance of the dissociation in the unfolding. The unfolding patterns obtained from the melting curves of each protein imply the existence of multiple intermediate states and/or processes. Our data provide evidence that the pretransition in the unfolding of ribonuclease S is due to partial unfolding of the S protein/S peptide complex and that the dissociation occurs at higher temperature. Our observations are consistent with a sequential denaturation mechanism in which at least one partial unfolding step comes before the main conformational transition, which is instead a concerted, final unfolding/dissociation step.     

The thermal unfolding and domain structure of Na+/K+-exchanging ATPase. A scanning calorimetry study.

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.     

Human RAD52 protein has extreme thermal stability.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1--192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2 degrees C. The RAD52(1--192) mutant had only two thermal transitions at 47.6 and 100.9 degrees C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.