Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride.

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.     

Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.     

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.     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.     

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

3-磷酸甘油醛脱氢酶胍变性时的活力及构象变化

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中的内源荧光及剩余活力的变化结果提示:apo酶及holo酶的活力在胍浓度为0.5M左右可完全丧失.同时伴有内源荧光强度的下降,光谱宽度的增加和335nm最大发射峰的红移(提示了色氨酸残基的暴露).与已经报导的肌肉酶(内源荧光强度在胍浓度为0.4—1.2M范围相对稳定)不同,酵母酶内源荧光在此浓度范围内表现为逐渐降低.在0.7M胍溶液中,内源荧光变化动力学过程只能测出一相,而酶失活动力学过程为快慢两相,快相动力学速度常数至少大于内源荧光降低速度常数三个数量级以上.以上结果提示:低浓度胍可引起该酶的完全失活,活性部位的空间构象比酶分子的构象更易受到变性剂的扰乱;有一个色氨酸残基位于或靠近酶的活性部位.     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.     

FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: "hidden" intermediates at low denaturant concentrations

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.     

3-磷酸甘油醛脱氢酶的胍变性——全位与半位修饰酶的比较

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中内源荧光及NAD荧光衍生物的410nm特征荧光发射光谱的变化结果提示,全位及半位修饰羧甲基酶活性部位与NAD共价连接的荧光衍生物的形成,明显受到盐酸胍的干扰,并且前者比后者更为显著.全位及半位修饰光照酶的特征荧光在低胍浓度下较内源荧光降低更为显著,同时伴有最大发射峰先红移后兰移的现象.NAD荧光衍生物的特征荧光在胍溶液中减弱的动力学过程分为快相和慢相,快相一级动力学常数比慢相的大两个数量级,全位及半位修饰酶的特征荧光的减弱快慢相速度常数分别属于同一个数量级.以上结果提示:酶活性部位的构象较整个分子来说更易被变性剂扰乱,柔性强于整个分子;NAD荧光衍生物的形成需要活性部位具有正确的空间几何结构.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

NMR studies on the induced denaturation of lysozyme by guanidine hydrochloride in the presence of the inhibitor (NAG)3

The stabilization of the folded conformation of lysozyme, arising from the binding of the inhibitor (NAG)3 against induced denaturation, is demonstrated from the 1H-nmr spectra of the enzyme. The nmr spectra reveal that the binding of the denaturant (GuHCl) to the enzyme is associated with changes in the conformation of the enzyme. The binding site of the inhibitor site C also serves as one of the binding sites of GuHCl. The observation that higher denaturant concentrations are required in the unfolding of Lys-(NAG)3 as compared to free Lys can be explained partly in terms of the existence of a competitive binding to the enzyme involving the (NAG)3 and GuHCl molecules.     

Guanidinium chloride-induced spectral perturbations of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid confound interpretation of data on molten globule states

We describe limitations in the use of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) to examine unfolding intermediates associated with guanidinium chloride (GuHCl)-induced protein denaturation. Several studies have used alterations in fluorescence emission of bis-ANS to quantify the population of “molten globule” states. Our findings indicate that the observed changes in bis-ANS spectroscopic properties could originate from the interactions of bis-ANS and GuHCl and the aggregation of the dye at higher GuHCl concentrations. We posit that in the absence of additional complementary structural or spectroscopic measurements, the use of bis-ANS emission alone to monitor protein conformations can be misleading.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

活性部位的柔性

比较酶在变性过程中构象和活力变化,发现在活性完全丧失时尚无可察 觉的整体构象变化。排除变性剂抑制和寡聚酶解聚等可能性之后,提出了酶活性部位柔性假说。随后用多种实验方法直接证实了活性部位的构象变化先于分子整体构象变化,并与活性丧失同步,根据催化过程中活性部位构旬变化,以及限制活性部位构象变化对酶活性的影响,提出了酶活性部位柔性为酶充分表现其催化活性所必需的设想。