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Biomedical subjects

G Mei

Publications and source records attributed to G Mei.

36 records · Page 2Linked to original sources
PubMed1996

Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin.

Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this postsynthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly alpha-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a maximum of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, circular dichroism, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of >/=10% citrulline results in protein denaturation. Cyanate modification of the lysines in model alpha-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.

Amino Acid Sequence↗
PubMed1996

Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism.

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2, 4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the final sigma-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as kcat degrees /kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for Km GSH, while Km CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and Km GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and Km GSH, thereby suggesting a possible correlation between kcat, Km GSH, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of Km GSH, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 A occurring between helix 2 and helix 4 when GSH binds to the G-site.

Binding Sites↗
PubMed1996

A stochastic model for the sigmoidal behaviour of cooperative biological systems.

A stochastic model for cooperative transitions in biological systems based on a Markov chain is proposed. This model requires only two parameters, the mean probability, p, and the coupling capacity, Deltap, which measure the probability of forming a new weak bond depending on the number of similar bonds already formed and it is also responsible for the transition. In this paper we show how the model works for a large number of identical molecules and how it can be useful for studying the noise around the centre of the transition where, increasing the degree of cooperativity, i.e. the number n in the well-known Hill equation, the width of the noise increases along with its fractal dimension. A simple relationship between the degree of cooperativity and the parameter Deltap is proposed, suggesting that the cooperativity of real biological transitions is related to the coupling capacity Deltap of the present model.

Journal Article↗
PubMed1996

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).

Azurin↗
PubMed1995

Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no alpha or beta structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.

Amino Acid Sequence↗
PubMed1995

New polarized light microscope with precision universal compensator.

A new type of polarized light microscope ('new pol-scope') for fast and orientation-independent measurement of birefringent fine structure has been developed. The design of the new pol-scope incorporates a precision universal compensator made from two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at all points of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen. The sensitivity of the current instrument is 0.1 nm of specimen retardance, measured with data gathered in 0.43 s at all 640 x 480 image points. Examples of birefringence measurements in biological (microtubule arrays) and industrial (magneto-optical disc substrate) specimens are presented.

Microscopy, Polarization↗
PubMed1994

Biotin and biotin analogues specifically modify the fluorescence decay of avidin.

Avidin, a basic tetrameric glycoprotein, isolated from hen egg-white, binds up to four molecules of biotin with exceptionally high affinity. The presence of tryptophanyl residues in the active site pointed out the opportunity of correlating the protein fluorescence with biotin binding. We have performed both steady state and dynamic fluorescence experiments using biotin or biotin-derived molecules (biotinamine, diaminobiotin and iminobiotin) as ligands. The fluorescence decay data can only be fitted by two continuous distributions of lifetimes which may reflect the presence of static or dynamic microheterogeneity in the environment of the tryptophan residues. We observed that the binding of biotin, biotinamine and iminobiotin reduces the widths of both distributions to discrete lifetimes thus indicating a more homogenous environment for the emitting tryptophan residues. Instead, the binding of diaminobiotin, which lacks the imidazolone ring, affects one lifetime distribution only. The binding of biotin also affects the rotational correlation time of avidin, which becomes shorter, suggesting a more compact structure of the ligated protein. The utility of analyzing the fluorescence in terms of distributions appears to be further warranted.

Animals↗
PubMed1994

Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy.

Two mutants of the blue copper protein azurin from Pseudomonas aeruginosa, Ile7Ser and Phe110Ser, were prepared. The mutations were aimed at affecting the mobility and the fluorescence properties of Trp48, the only tryptophan residue present, which in the wild-type protein is located in a highly hydrophobic and rigid environment. EPR, UV-vis, and NMR spectroscopy show that the copper binding site and the overall structure of the wild-type protein are preserved and that structural effects occur only on a local scale. Steady-state fluorescence spectra of both mutants, particularly in the copper-free form, show that tryptophan fluorescence is dramatically affected by the introduction of a polar residue close to it. The emission maximum is red-shifted and dependent on the excitation wavelength. This indicates a loosening of the matrix around the indolyl side chain and an increase of the effective dielectric constant of the microenvironment. Time-resolved fluorescence spectroscopy also shows substantial changes in the fluorescence lifetimes and in the distribution of the lifetimes of the mutants; these variations are interpreted in terms of a change in solvation of the Trp48 side chain.

Azurin↗
PubMed1993

Molten globule monomers in human superoxide dismutase.

The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microheterogeneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 7224-7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state.

Circular Dichroism↗
PubMed1993

Some pitfalls in the measurement of blood glutathione.

1. We report some pitfalls in the measurement of whole blood and plasma glutathione in man. 2. Using a simple tourniquet to the forearm and a 21-gauge needle, blood samples were collected by brachial vein puncture from healthy subjects. Whole blood and plasma were analysed for total glutathione, including the reduced and oxidized forms, by a spectrophotometric recycling method involving the glutathione reductase/NADPH couple. 3. The concentration of oxidized glutathione was determined after treatment of aliquots with either 2-vinylpyridine or N-ethylmaleimide to trap reduced glutathione. Reduced glutathione in the native samples could then be obtained by subtraction. 4. When the reagents were added to separated plasma, 2-vinylpyridine yielded values for oxidized glutathione that were twice as high as with N-ethylmaleimide. In whole blood studies the discrepancy was even greater, and the problem was not resolved by deproteinization of samples with 5-sulphosalicylic acid. Using N-ethylmaleimide, levels of oxidized glutathione were less than 1% of total glutathione in whole blood. 5. Despite attempts to minimize haemolysis, lysed erythrocytes contributed on average 25% to the 'plasma' glutathione concentration.

Blood Specimen Collection↗
PubMed1992

Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study.

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoenzymes↗
PubMed1991

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine.

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.

Affinity Labels↗
PubMed1991

Intrinsic fluorescence of the bacterial copper-containing protein amicyanin.

The fluorescence properties of the single tryptophanyl residue present in amicyanin from Thiobacillus versutus are very similar to those of azurin from Pseudomonas aeruginosa and other mononuclear blue copper proteins. The emission maximum is well structured and centered at 318 nm. The quantum yield is strongly affected by the presence of copper, the removal of which is accompanied by a more than sixfold increase in fluorescence, without change in shape. The fluorescence decay of holo-amicyanin is heterogeneous with a longer component of 5.7 ns and a shorter one of 0.7 ns accounting for 90% of the total emitting molecules. Copper-free amicyanin shows instead a single exponential decay (3.3 ns) of intrinsic fluorescence. This lifetime decreases as the temperature increases as does the longer lifetime component of holoamicyanin.

Bacterial Proteins↗
PubMed1990

Dynamic fluorescence in copper proteins. Selected examples.

The fluorescence properties of three copper proteins, namely human superoxide dismutase, Pseudomonas aeruginosa azurin and Thiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.

Azurin↗
PubMed1990

A time-resolved fluorescence study of human copper-zinc superoxide dismutase.

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.

Humans↗
PubMed1990

Fluorescence lifetime distributions in human superoxide dismutase. Effect of temperature and denaturation.

The internal dynamics of human superoxide dismutase has been studied using time-resolved fluorescence. The fluorescence decay has been analyzed using continuous distribution of lifetime values. The effect of temperature and conformational state on the lifetime distribution has been investigated. The emission of the single tryptophan residue depends on the nature and dynamics of the protein matrix. Conformational changes have been induced by increased concentration of guanidinium hydrochloride. We found that both temperature and conformation strongly effect the width of the lifetime distribution.

Biophysical Phenomena↗
PubMed1989

Time-resolved extrinsic fluorescence of aromatic-L-amino-acid decarboxylase.

The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.

Animals↗