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W Pfeil

Publications and source records attributed to W Pfeil.

At least 37 records · Page 2Linked to original sources
PubMed1993

Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast.

The temperature-dependent conformational equilibrium of 3-phosphoglycerate kinase has been studied in the temperature range from 1 to 30 degrees C by means of dynamic light scattering, small-angle X-ray scattering, differential scanning calorimetry, circular dichroism spectroscopy, and fluorescence spectroscopy. At 28 degrees C and in the presence of 0.7 M guanidine hydrochloride (GuHCl), the radius of gyration (RG) and the Stokes radius (RS) are 2.44 and 3.09 nm, respectively. Decreasing the temperature effects unfolding of the molecule, a process that involves two stages. The two stages correspond to the successive unfolding of the N-terminal and C-terminal domains. The peak maxima of the excess heat capacity, determined from differential calorimetric scans, extrapolated to 0 scan rate, are positioned at 16.5 degrees C for the N-terminal domain and at 6.3 degrees C for the C-terminal domain. At 4.5 degrees C, the radius of gyration and the Stokes radius increase to 7.8 and 4.8 nm, respectively. The persistence length and the length of the statistical chain segment of the unfolded polypeptide chain are 1.74 and 3.48 nm, corresponding to five and ten amino acids, respectively. At 1 degrees C, the dimensions of the unfolded chain nearly agree with the predicted dimensions under theta conditions. Thus, the conformational changes upon cold denaturation can be described by a transition from a compactly folded molecule to a random coil. The conformation-dependent ratio rho = RGRS-1 increases from rho = 0.79 to rho = 1.63. The volume of the unfolded chain is 30 times larger than that of the folded chain in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1993

Thermodynamics of apocytochrome b5 unfolding.

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).

Animals↗
PubMed1992

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.

Amino Acid Sequence↗
PubMed1992

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.

Amino Acids↗
PubMed1989

Microcalorimetric studies of conformational transitions of ferricytochrome c in acidic solution.

The conformational transitions of ferricytochrome c in acidic solutions with different NaCl concentrations have been studied by scanning and isothermal microcalorimetry. It is shown that ferricytochrome c adopts three different forms which are realized under the considered conditions: native, denatured (unfolded) and a compact native-like form with unique tertiary structure. The thermodynamic parameters of the corresponding transitions have been measured and the changes in the number of bound ligands (H+ and Cl-), accompanying these transitions, have been determined by analyzing the temperature, pH and ionic strength dependence of these parameters.

Calorimetry↗
PubMed1987

Is thermally denatured protein unfolded? The example of alpha-lactalbumin.

The degree of unfolding of various forms of human and bovine alpha-lactalbumin was characterized by the chromatographically determined distribution coefficient. Molecular size increases as follows: native protein approximately equal to apo form less than acid form much less than thermally unfolded form approximately equal to guanidine hydrochloride-unfolded protein.

Animals↗
PubMed1986

Lactoperoxidase consists of domains: a scanning calorimetric study.

Thermal unfolding of lactoperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was studied by means of differential scanning calorimetry and optical methods. The protein consists of at least two domains differing in thermostability. The prosthetic group belongs to the domain of lower thermostability. Thermodynamic parameters of protein unfolding are given and found to be similar to corresponding data for globular proteins.

Animals↗
PubMed1986

Physical nature of the phase transition in globular proteins. Calorimetric study of human alpha-lactalbumin.

The guanidine hydrochloride-induced unfolding of human alpha-lactalbumin has been studied by isothermal calorimetry. It has been shown that a cooperative transition takes place only in the concentration interval of the denaturant between 0.3 and 2 mol X l-1. The cooperative transition coincides with the transition detected by circular dichroism in the near-ultraviolet region which reflects the destruction of the specific environment of aromatic side groups. According to scanning calorimetric investigations, the transition disappears in the acid form of the protein where circular dichroism of aromatic side groups is practically absent. At higher concentrations of guanidine hydrochloride, where destruction of the secondary structure and unfolding of the chain are observed, there is no cooperative heat absorption.

Calorimetry↗
PubMed1983

Thermodynamic investigations of cytochrome b5 unfolding. II. Detergent-solubilized cytochrome b5 in solution and in a reconstituted system with dimyristoyl phosphatidylcholine.

Thermal unfolding of the detergent-solubilized cytochrome b5 was investigated by scanning calorimetry. The protein shows different thermostability in the presence and absence of detergent, and it achieves the maximal transition temperature after incorporation into dimyristoyl phosphatidylcholine liposomes. However, transition temperature and Gibbs energy change at unfolding are still lower than that of the tryptic fragment of cytochrome b5 in aqueous solution. Cytochrome b5 undergoes in aqueous solution in the absence of detergent an irreversible, complicated transition, but it remains in the associated state after thermal denaturation. Half transition temperature, enthalpy and heat capacity changes of cytochrome b5 unfolding under various external conditions are reported and compared with the corresponding values of the tryptic fragment of the protein. The thermodynamic data and independent results are suitable for detailing a model proposed by Tanford (The Hydrophobic Effect (1980), pp. 205-211, John Wiley & Sons, New York) for the spatial arrangement of the protein within the membrane.

Animals↗
PubMed1983

Statistical thermodynamical treatment of protein unfolding.

The statistical thermodynamical model of protein structure according to Ikegami (Biophys. Chem. 6, 117 (1977] has been applied to scanning calorimetric investigations of nine globular proteins. The proteins show unique properties with respect to bond energy, chain entropy and cooperative energy but individual differences with respect to the number of noncovalent bonds and a hydrophobicity parameter. The parameters specifying protein structure are correlated with experimentally determined heat capacity changes. The treatment is suited for prediction of structural parameters as shown by the alpha-lactalbumin example.

Calorimetry, Differential Scanning↗
PubMed1983

[The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450].

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.

Animals↗
PubMed1982

Different modes of membrane interactions of the signal sequence of carp preproinsulin and of the insertion sequence of rabbit cytochrome b5.

The signal segment of the secretory protein carp preproinsulin is shown to be bound by a protein receptor present in the rough endoplasmic reticulum membrane. The receptor does not bind the insertion segment of the integral membrane protein cytochrome b5. On the other hand, the insertion sequence, in contrast to the signal sequence, is dissolved into the lipid bilayer of natural and artificial membranes. Hydrophobicity or length per se of the two types of peptides cannot be responsible for their different behaviour. We rather propose that the difference resides in their tertiary structure. Insertion peptides may form a compact structure with a diffuse hydrophobic surface, presumably by internal hydrogen bonding. Signal peptides would form hydrogen bonds with a membrane-bound receptor protein, presumably by producing a beta-sheet structure, but their extended structure in aqueous solution would not allow them to dissolve into lipid bilayers.

Amino Acid Sequence↗
PubMed1982

Role of the hydrophobic tail of cytochrome b5 in the interaction with cytochrome P-450 LM2.

The interaction of cytochrome P-450 LM2 with cytochrome b5 is accompanied by a high spin shift in P-450 LM2 and the improvement of a second derivative spectra in the near ultraviolet region. After incorporation into phospholipid vesicles the interaction between P-450 LM2 and b5 is increased according to a decrease of the apparent binding constant. The involvement of a tryptophanyl residue in the interaction will be discussed. Contrary the tryptic fragment of cytochrome b5 which lacks the membrane binding tail does not show an interaction with P-450 LM2 either in the absence or presence of phospholipids.

Animals↗
PubMed1981

The problem of the stability globular proteins.

The article gives a survey on protein stability. Starting out from approaches for stability measurement which are based on the determination of Gibbs energy change in protein unfolding by denaturants, protonation, heat, scanning calorimetry, and hydrogen exchange, their implications such as reversibility, completeness of unfolding and the two-state assumption are dealt with. A data compilation of Gibbs energy change in unfolding of different proteins is given. The data, which for the most part range between 25 and 60 kJ mol-1 are discussed in terms of protein functioning, turnover, and structural properties. Phase diagrams are proposed in order to realize a more comprehensive thermodynamic treatment of proteins. Factors which contribute to protein stability are summarized. The paper includes the thermodynamic principles of protein stability as well as special studies on proteolytic fragments, amino acid replacements, cross links, prosthetic groups, and ions which contribute to protein stability.

Animals↗
PubMed1981

Thermodynamics of alpha-lactalbumin unfolding.

Thermodynamic investigations of alpha-lactalbumin have been performed by isothermal calorimetric guanidine hydrochloride titrations as well as by scanning calorimetric measurements in the presence and absence of guanidine hydrochloride. Compared with lysozyme, alpha-lactalbumin is less stable, and its changes of enthalpy and heat capacity at unfolding are lower. Thermal unfolding of alpha lactalbumin can be described to the first approximation by the two-state transition model even in the presence of guanidine hydrochloride.

Animals↗