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B Alpert

Publications and source records attributed to B Alpert.

At least 19 recordsLinked to original sources
PubMed1999

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

Animals↗
PubMed1999

Heterogeneous motions within human apohemoglobin.

Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 degrees C using CD spectra in the far-UV (200-250 nm) and near-UV (250-300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located in the heme cavities. The long decay time of the dye emission (> 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (congruent with 3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 degrees C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the alpha14, beta15 and beta37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.

Adult↗
PubMed1998

Solvent effects on horse apomyoglobin dynamics.

The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity of the two Trp residues, without inducing particular constraints on these very residues. Formamide increases, in the same way, the polarity and the protein flexibility while ethanol reduces both. The present fluorescence work also shows that, whatever the outside solvent, the two Trp residues W7 and W14, embedded in the A, G, and H helix core, are equally and statistically reached by small molecules diffusing inside the protein matrix. Hydrogen-tritium exchange measurements on the protein in mixed solvents reveal that the dynamics of the A, G, and H helix cluster and of the B and E helixes are greatly influenced by the nature of the outside medium. A small amount of formamide in the buffer increases the protein fluctuations while an ethanol-water mixture reduces them. We suggest that the hydratation state of the protein surface could be the relevant parameter of the protein dynamics.

Animals↗
PubMed1998

Dynamic and structural properties of glucose oxidase enzyme.

The catalytic oxidation of beta-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I'band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of alpha-helix present in the protein outer shell, but reorganizes a fraction of random coil to beta-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

Aspergillus niger↗
PubMed1998

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.

Animals↗
PubMed1998

Hypertension in young patients after renal transplantation: ambulatory blood pressure monitoring versus casual blood pressure.

The results of ambulatory blood pressure monitoring (ABPM) in children after kidney transplant were analyzed to ascertain any alteration in circadian BP profile, degree of hypertension, and efficacy of therapy. The data were also compared with casual BP data and left ventricular mass index (LVMI). We have examined 30 patients (17 male, 13 female), mean age 16.1+/-3.6 years after kidney transplant. All patients were receiving triple immune-suppressive therapy and 20 of them were also taking antihypertensive therapy. They underwent clinical examination with measurement of BP at rest, echocardiogram mono-2D, and ABPM. The following ABPM parameters were recorded: systolic (S) and diastolic (D) BP at rest; mean 24-h SBP and DBP; mean daytime SBP and DBP; mean nighttime SBP and DBP; nocturnal fall in SBP and DBP; and mean daytime and nighttime heart rate (HR). The patients were divided in two groups. Group A consisted of 20 patients taking antihypertensive treatment; group B consisted of 10 patients not taking antihypertensive treatment. Casual and ABPM data for the two groups were compared using the Student t test for unpaired data. Blood pressure at rest and LVMI were not statistically different between the two patient groups. The ABPM data showed statistical differences between the two groups for mean 24-h SBP and DBP, daytime and nighttime SBP, nighttime DBP, fall in nocturnal DBP, and nighttime HR. Mean 24-h SBP and DBP, mean daytime SBP and DBP, and mean nighttime SBP and DBP were significantly correlated to LVMI (respectively, P = .009, P = .005, P = .008, P = .007, P = .05, and P = .01). Twenty-four-hour ABPM was more useful in the diagnosis and management of hypertension than was casual BP at rest.

Adolescent↗
PubMed1998

Open reduction and internal fixation of acute mandibular fractures in adults.

The mandible is often fractured during maxillofacial trauma because of its prominence within the facial skeleton and the fact that it is a favorite target in interpersonal violence. Treatment of mandibular fractures has made significant advances over the years due to the study of biomechanical principles, advancements in biomaterials and instrumentation and scientifically based research of treatment outcomes. This article will review the principles and philosophy of AO/ASIF techniques as applied to treatment of mandible fractures. Representative cases of different mandible fractures and their treatment will reinforce the clinical application of rigid fixation techniques.

Adult↗
PubMed1996

Probing pH and pressure effects on the apomyoglobin heme pocket with the 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid fluorophore.

The environmentally sensitive fluorophore 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid (DANCA) has been used to probe the apomyoglobin heme pocket. The unexpected polarity of this domain is generally interpreted as arising from dynamic dipolar relaxation of the peptide dipoles surrounding the heme pocket. In the present work we reexamine the photophysical properties of DANCA in a variety of solvents and complexed with apomyoglobin (apoMb) to further probe the heme pocket environment as a function of external solvent conditions. Absorption and excitation spectra in a number of solvents are consistent with the well-known pi*<--pi (LE) and pi*<--n (CT) electronic absorption transitions observed for naphthylamine derivatives. Dual emission is also a well-documented property of such derivatives. Based on the time scale of the heterogeneity in the decay of the DANCA fluorophore observed in a series of solvents, we propose that the emission properties of DANCA in apoMb are not uniquely attributable to dynamic relaxation events, but also reflect dual emission from both a long-lived, red CT state and the shorter-lived, blue LE state. The pH studies in the range of pH 5-9 of the emission properties of DANCA in apoMb support this hypothesis. They also suggest a specific interaction of DANCA with one or both of the pocket histidyl residues, which leads to a drastic static quenching and red shift of the bound DANCA fluorescence upon protonation. Similar effects are observed with increasing pressure, indicating that these two perturbations alter the DANCA-apoMb complex in a similar fashion. The pressure-induced form of the protein is distinct both energetically and structurally from the previously characterized acid intermediate, in that it is populated above pH 5 and retains a significant degree of integrity of the heme pocket.

Animals↗
PubMed1995

Solvent-induced structural distortions of horse metmyoglobin.

Structural and dynamic constraints produced by the surrounding solvent on the aquometmyoglobin molecule were investigated by means of circular dichroism and Fourier-transform infrared spectroscopies, tritium/hydrogen exchange kinetics and small-angle neutron-scattering experiments. Formamide and ethanol were chosen as cosolvents because they are known to increase and decrease protein activity, respectively. The CD measurements in the Soret region show that no changes occur in the heme molecular structure nor in the protein near the heme. The results of proton-exchange kinetics experiments indicate that the conformational dynamics of aquometmyoglobin is only marginally affected by the cosolvents. However, the small-angle neutron-scattering spectra strongly suggest that these cosolvents induce some distortions of the tertiary conformation. According to the ultraviolet CD and Fourier-transform infrared data, the alteration of the tertiary conformation results from changes in both the number of intrachain hydrogen bonds and the structures of beta turns of type I' for formamide and of type II for either of the two cosolvents. The use of several techniques allows the present approach to demonstrates that the myoglobin structure is extremely sensitive to its environmental conditions.

Animals↗
PubMed1994

The heme iron coordination complex in His64(E7)Tyr recombinant sperm whale myoglobin.

By using site-directed mutagenesis of recombinant sperm whale (SW) myoglobin, the native distal histidine residue, at position 64 (the helical position E7), has been replaced with a tyrosine. The mutation of His64Tyr SW myoglobin has an analogous heme iron electronic structure as that of native hemoglobins M Boston and M Saskatoon. Optical spectroscopy showed that the distal tyrosine bound to the heme iron had a pK value of 5.6. In the pH range of 4.7-11.0, electron spin resonance spectroscopy suggested the presence of two heme iron ligation schemes: the heme iron bound to a distal water molecule or to a distal tyrosine residue. The heme iron coordination in the wild-type myoglobin and in the His64Tyr SW Mb mutant was studied by X-ray absorption near-edge structure (XANES) spectroscopy. Indeed, the heme iron K-edge reflects the electronic organization of the metal inside the six-coordinated complex. Comparative analysis of X-ray absorption heme iron K-edge shapes showed that the heme iron of His64Tyr SW myoglobin is bound to the oxygen atom from the phenol group of the distal tyrosine residue (Fe-OH phi). When the pH value decreased from pH 7 to 5.6, the Fe-OH phi bond strength decreased, resulting in an increase of the heme iron high-spin population of His64Tyr SW myoglobin. At low pH values, the Fe-OH phi bond can be disrupted with the possibility of heme iron binding of another ligand having a higher affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

An infrared study of 2H-bond variation in myoglobin revealed by high pressure.

A high-pressure Fourier-transform infrared technique was used to probe the evolution of 2H bonds inside the helical segments of myoglobin in relation to p2H, Tris concentration in the medium and iron-ligand nature. The analysis was focused on changes in the conformation-sensitive amide-I' band, reflecting the peptide C = O group stretching vibrations coupled to the in-plane N-2H bending and C = N stretching modes. From data obtained under high pressure, the strength of 2H bonds, inside the alpha-helical segments of the protein at atmospheric pressure, is not simply a function of p2H and salt concentration. At low Tris concentration (50 mM), the strength of these 2H bonds increases with p2H, whereas for a higher Tris concentration (100 mM) this strength is lower at p2H 7 than at p2H 6.0 or 8.5. It is also observed that the azidometmyoglobin molecule exhibits tighter intrahelical interactions and lower sensitivity to pressure than aquametmyoglobin. Information is also presented regarding interhelical interactions in relation to the solvent.

Animals↗
PubMed1992

Experience with rigid fixation of mandibular fractures and immediate function.

The results of using rigid fixation for 75 mandibular fractures in 52 patients and allowing immediate function were retrospectively analyzed. Sixteen percent of fractures developed postoperative infection. All infections were in fractures associated with teeth; none occurred in fractures fixed within 24 hours of injury. Angle and posterior body fractures were most likely to become infected. Fractures with improperly applied rigid fixation also were subject to a high infection rate. It was unusual for infection involving this type of treatment to prolong the course of therapy or affect the final outcome.

Adolescent↗