Jun-Mei Zhou

Unraveling multistate unfolding of rabbit muscle creatine kinase.

GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of phase diagram, i.e. I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are fluorescence intensity values measured on wavelengths lambda(1) and lambda(2) under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated.

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.

Inactivation precedes conformation change during thermal denaturation of adenylate kinase

During the thermal denaturation of rabbit muscle adenylate kinase, the extents and rates of both unfolding and aggregation are dependent on protein concentration. Under identical conditions, inactivation takes place at a lower temperature than noticeable conformational changes and aggregation as measured by fluorescence, second derivative absorption spectroscopy, far ultraviolet circular dichroism and light scattering. Kinetics of inactivation can be resolved into two phases and at the same protein concentrations, the unfolding and aggregation rates are about one order of magnitude slower than the fast phase and approximately the same as the slow phase rate of the inactivation reaction between 35 and 60 degrees C. This is in general accord with the suggestion made previously that the active site of this enzyme is situated in a region more flexible than the molecule as a whole (Tsou, C.L. (1986) Trends Biochem. Sci. 11, 427-429). The inactivated enzyme cannot be reactivated by cooling and standing at 4 degrees C but can be over 80% reactivated by cooling and first standing in 3 M guanidine hydrochloride followed by diluting out the denaturant.

COMPARISON OF INACTIVATION AND CONFORMATIONAL CHANGES OF D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE DURING THERMAL DENATURATION

The inactivation of D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5 degrees C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5 degrees C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427).

FTIR studies of secondary structures of bovine insulin and its derivatives

The amide I bands of the deconvolved FTIR spectrum of bovine insulin, despentapeptide (B26-B30) insulin and desoctapeptide (B23-B30) insulin in D2O solution have been assigned to alpha-helix, the 3(10) helix, irregular helix, extended chains, beta-turns and other secondary structures. From the peak areas the relative contents of these structures obtained are in general agreement with those calculated from the known structures of porcine insulin and DPI in the crystalline state. The main difference in the structure of DOI with those of insulin and DPI is the shortening of the helix segment and an extended chain for the C terminal segment in the B chain.

Activation of chicken liver dihydrofolate reductase in concentrated urea solutions

The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.

Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride

The unfolding of adenylate kinase in GuHCl of increasing concentrations has been followed by a combination of different methods. Molecular packing was measured by size-exclusion chromatography (SEC), exposure of buried Tyr residues by second- derivative spectra, loss of secondary structure by circular dichroism in the far-ultraviolet and the decrease in surface hydrophobicity by ANS binding. The conformational changes of adenylate kinase as followed by the above methods depend differently on GuHCl concentration. The concentrations of GuHCl at which 50% changes as measured by the above four methods occur are 0.3, 0.46, 0.64 and 0.64 M, respectively. SEC measurements show that with increasing GuHCl concentrations, the process of unfolding of adenylate kinase involves two slowly interconvertible intermediate stages, I1, and I2, the last is in a more advanced state of unfolding but is still more compact than the fully unfolded state, U, as indicated by their elution volumes in the SEC profile. There is also evidence to suggest that both the intermediates I1 and I2 may contain additional intermediary components in rapid equilibrium as indicated by the gradual shift of both peaks in the SEC elution profile. A sequential mechanism is suggested for the unfolding of adenylate kinase with increasing guanidine hydrochloride concentrations.

Spin-labeling probe on conformational change at the active sites of creatine kinase during denaturation by guanidine hydrochloride

The conformational change at the active sites of creatine kinase and its protection by substrates during guanidine denaturation were investigated by monitoring the ESR spectra of the nitroxide radical covalently bound to the reactive thiols of the enzyme. For the enzyme undenatured (pH 9.0) and in the presence of low concentrations of guanidine, i.e. less than 1 M, there are two kinds of enzyme molecule, one of which is bearing a compact structure at the active site and the other is of a looser structure. The content of the latter increases with increasing denaturant concentration. At concentrations of guanidine hydrochloride higher than 1 M, the structure of the enzyme molecule is monomorphic and becomes looser and looser with an increase of guanidine hydrochloride concentration. The existence of a nucleotide substrate complex protects the structure at the active sites of the enzyme from being changed, up to a concentration of denaturant of 0.2 M, while creatine has no protective effect.

Conformational specificity of trigger factor for the folding intermediates of α-lactalbumin

Abstract To understand the structural features of polypeptides recognized by trigger factor, a number of conformational derivatives of αLA were prepared and their effects on the trigger factor assisted refolding of GAPDH were investigated. It was found that the conformers of αLA that efficiently reduce the trigger factor assisted reactivation of guanidine-denatured GAPDH by competitively binding with trigger factor are those derivatives that have loose structure. This suggests that trigger factor binds mainly to intermediates formed early during folding of GAPDH.

Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5'-phosphate.

The alpha-amino group of Lys-1 and the epsilon-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5-phosphate (PLP) separately. The effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decreased obviously during denaturation by low concentrations of GdnHCl indicating conformational change of the active site is a parallel event to the inactivation of the enzyme. Time-correlated fluorescence lifetime measurements revealed the existence of two conformational states of PLP-modified RNase A and a shift of the conformation in favor of the slow-decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole.