Orientation of fusion-active synthetic peptides in phospholipid bilayers: determination by Fourier transform infrared spectroscopy

A group of synthetic peptides having an amino acid sequence related to the N-terminal region of the influenza virus hemagglutinin HA-2 chain can induce phospholipid membrane fusion in a pH-dependent manner. These peptides bind to membranes to form alpha-helices even at pH's where no fusion activity is seen. We determined the orientation of these alpha-helical peptides in lipid multibilayers using attenuated total reflection infrared spectroscopy and found that the peptide alpha-helices took a preferential orientation, the helix axis being about 70 degrees from the normal of the membrane plane, or in other words rather parallel to the membrane plane. The orientation was almost independent of pH and a modification of the N-terminal amino group which reduced the fusion activity of the peptides. The determination was carried out for peptides in lipid multibilayers in dry or hydrated (membranes equilibrated with D2O vapor) conditions. Although a slight decrease in the helix orientation angle from the membrane normal was noticed for a hydrated system, the difference between the results for dry and hydrated conditions was small.     

Fourier transform infrared spectroscopic study of ion binding and intramolecular interactions in the polar head of digalactosyldiacylglycerol

The study describes the changes in basic hemodynamic parameters after long-term antihypertensive therapy with cilazapril--a new ACE inhibitor lacking a sulfhydryl group--in hypertensive patients and the drug effects on renal function, glucose tolerance and lipid metabolism. 30 patients (18 males, 12 females, mean age: 53.3 +/- 18 years) with mild to moderate essential hypertension were studied. The following determinations were performed in patients, before and after 4.5 months of cilazapril monotherapy at a dose of 5 mg/24 h: (a) antihypertensive action of the drug (arterial pressure at rest and during a 24-hour recording); drug effects on left ventricular (LV) mass index; its contractility indexes (%FS, EF) and the left atrial emptying index were studied by means of echocardiography; (b) plasma insulin concentration during oral glucose tolerance tests, in the fasting state, after the administration of 75 g glucose per os, as well as the changes in the insulinogenic index and the 6-keto-PGF1 alpha/TXB2 ratio, and (c) drug effect on renal function (urea, creatinine, uric acid, plasma electrolytes), blood lipid profile (total cholesterol, triglycerides, HDL-CH) and serum transaminases. Long-term drug administration exhibits an effective antihypertensive action, without causing reflex tachycardia and also reduces the LV mass index without affecting its EF, while improving its diastolic function. It does not significantly affect the various biochemical parameters, and achieves glucose regulation, both in the fasting state and after glucose loading, with a decrease in the insulinogenic index, and simultaneously increases the 6-keto-PGF1 alpha/TXB2 ratio. The existence of a direct cause-effect relationship between the changes in the above hormone systems is possible.     

A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in solution

An attenuated total reflectance Fourier transform infrared method has been developed that allows collection of spectra from proteins in solution. This method eliminates any structural perturbations induced by the internal reflection element (IRE), and thus the spectra reflect the solution conformation of the protein. A key feature of the method is subtraction of the signal from any protein adsorbed to the IRE. The advantages of this method include the small amount of sample required and the high sampling rate. Attenuated total reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) is more versatile than transmission FTIR because it is possible to collect spectra of nontransparent samples, to use samples of very low protein concentration (< or = 0.3 mg/ml), and to study proteins in the presence of strongly absorbing solutes (such as denaturants). The experimental procedures and data processing routines developed were evaluated by collecting spectra from a set of 13 proteins and evaluating their accuracy with a partial least-squares analysis. The relative mean and standard deviation errors for the basis set analysis were 6.3% for alpha-helix, 5.9% for beta-sheet/extended structure, and 4.4% for turn, which are similar to values from comparable analyses of transmission FTIR spectra. In addition, a detailed comparison between this solution ATR method and the hydrated thin-film ATR technique is presented.     

Predictions of protein secondary structures using factor analysis on Fourier transform infrared spectra: effect of Fourier self-deconvolution of the amide I and amide II bands

AAPB and its membership are faced with a number of giant challenges, including but not limited to: (1) the cost savings efforts of third-party payors and managed care organizations; (2) the lack of public awareness of biofeedback and its usefulness; and (3) the lack of sufficient research data on both the effectiveness and efficacy of biofeedback. In spite of these challenges, there are windows of opportunity that have been or which could be created to move biofeedback further into the realm of conventional treatment. We must focus our efforts on working together to: (1) create strategic plans for creating the future of applied psychophysiology and biofeedback; (2) educate all decision makers, including the general public; (3) establish better relationships with other professionals with common interest; (4) conduct more efficacy and effectiveness research; and (5) create a demand for our services so that the public will be more willing to pay for our services "out of their own pocket." In order for this to happen, we must stop fighting with each other and direct our energies to productive activities that can change fantasies into realities.     

Alteration of an intersubunit contact in hemoglobin variants: comparative study of modifications at position alpha 126 Asp (H9)

Four human hemoglobin variants have already been described at position alpha 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (-->Tyr), Hb Tarrant (-->Asn), Hb Fukutomi (-->Val), Hb Sassari (-->His). An additional variant, Hb West One (alpha 126 (H9) Asp-->Gly) is herein described. Aspartate alpha 126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the alpha-chains and of the C-helix of the beta-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these alpha 126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific alpha-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the alpha 126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur.     

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.     

Functional heterogeneity of the alpha and beta subunits in the association reaction between hemoglobin and carbon monoxide

A technique is described for the rapid inactivation and removal of excess ferricyanide used for the non-cryogenic oxidation of the unliganded subunits of the intermediates in the association reaction between hemoglobin and carbon monoxide. Under these conditions the asymmetric oxidized intermediates, which dissociate into non-identical dimers, disproportionate into their parent tetramers and four species, Hb+, HbCO, alpha 2+ beta 2CO, alpha 2CO beta 2+, are isolated by non-cryogenic isoelectric focusing. The relative concentrations of species alpha 2CO beta 2+ and alpha 2+ beta 2CO measure the overall distribution of the ligand between the alpha and beta subunits in the association reaction. At 20 degrees C in 0.1 M KCl, pH 7, preferential CO binding to the beta subunits was observed, in agreement with observations made by the cryogenic technique for the isolation of the intermediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem. 267 (1992) 8744].     

Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. All of the variants still retained significant activity, and all could be purified to homogeneity. FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme. There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant. This frequency is appropriate to a cysteine S-H stretching mode. It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized. Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain). The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme. The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 [Baburina et al. (1994) Biochemistry 33, 5630-5635] is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase. This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid.     

Motions in hemoglobin studied by normal mode analysis and energy minimization: evidence for the existence of tertiary T-like, quaternary R-like intermediate structures

The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like.     

Influence of ADP, AMP-PNP and of depletion of nucleotides on the structural properties of F1ATPase: a Fourier transform infrared spectroscopic study

Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.     

Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine

Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

Multiple modulation pathways of calcium channel activity by a beta subunit. Direct evidence of beta subunit participation in membrane trafficking of the alpha1C subunit

In order to study the precise mechanisms of alpha1 subunit modulation by an auxiliary beta subunit of voltage-dependent calcium channels, a recombinant beta3 subunit fusion protein was produced and introduced into oocytes that express the human alpha1C subunit. Injection of the beta3 subunit protein rapidly modulated the current kinetics and voltage dependence of activation, whereas massive augmentation of peak current amplitude occurred over a longer time scale. Consistent with the latter, a severalfold increase in the amount of the alpha1C subunit in the plasma membrane was detected by quantitative confocal laser-scanning microscopy after beta3 subunit injection. Pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase inhibitor, abolished the increase of the alpha1C subunit in the plasma membrane, attenuated current increase, but did not affect the modulation of current kinetics and voltage dependence by the beta3 subunit. These results provide clear evidence that the beta subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the alpha1 subunit function and the other is a chaperoning of the alpha1 subunit to the plasma membrane.     

Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.     

Peptides from the conserved ends of the rod domain of desmin disassemble intermediate filaments and reveal unexpected structural features: a circular dichroism, Fourier transform infrared, and electron microscopic study

Synthetic peptides representing the conserved ends of the rod domain of desmin are shown to disassemble preformed desmin filaments when added in moderate molar excess. This argues for a similar importance of both ends of the rod for filament stability. Recent structural models of intermediate filaments suggest close proximity of the ends and perhaps even an interaction (N. Geisler, J. Schünemann, and K. Weber, 1992, Eur. J. Biochem. 206, 841-852; P. M. Steinert, L. N. Marekov, R. D. B. Fraser, and D. A. D. Parry, 1993, J. Mol. Biol. 230, 436-452). Since the disassembling activity of the peptides, in addition to their sequences, should be related in some way to their secondary structure, we have investigated the structures of a number of related peptides which all arise from the ends of the rod using electron microscopic and spectroscopic methods. All peptides showed the expected alpha-helical structure at low concentrations in the presence of trifluoroethanol, as revealed by circular dichroism. At higher concentrations the peptides showed extensive self-aggregation into various types of filaments. The filaments contain the peptides in beta-sheet conformation as shown by Fourier transform infrared spectroscopy.     

Body temperature daily rhythms in the striped mouse Rhabdomys pumilio: the effects of alpha and beta blockade

Body temperature (Tb) daily rhythms and the effects of alpha and beta blockade were studied in the South African diurnal striped mouse Rhabdomys pumilio. Eleven mice (8 males and 3 females) with a body mass of 42.7+/-7.8 g (mean +/- SD) were tested. Mice were acclimated to a 13 h:11 h light-dark photoperiod at an ambient temperature of 25 degrees C. To assess the daily rhythm of pineal melatonin secretion, urinary 6-sulfatoxymelatonin (6-SMT) was determined. Mice displayed a robust Tb daily rhythm with an acrophase in the dark period, which is unexpected for a diurnal species. The nocturnal increase in Tb was accompanied by a significant rise in urinary 6-SMT. The beta blocker propranolol (4.5 mg/kg), injected 1 h before lights-off, resulted in a higher Tb value, whereas the alpha blocker prazosin (1 mg/kg) blocked the increase of Tb during the dark period. Prazosin also significantly attenuated the nocturnal increase of urinary 6-SMT. These results are in agreement with those obtained from the golden spiny mouse Acomys russatus and support the idea that small diurnal mammals retain the Tb rhythm of a nocturnal rodent. They also suggest that pineal melatonin secretion in these rodents is regulated by alpha rather than by beta receptors.