Interactions of parvalbumins with model phospholipid vesicles.

Interactions of Ca2+-binding proteins, parvalbumins, with model vesicles formed with both synthetic (dipalmitoylphosphatidylcholine) and natural (phosphatidylcholine and phosphatidylethanolamine) phospholipids have been revealed and studied by means of gel-chromatography, electron microscopy, intrinsic fluorescence and microcalorimetry methods. There are at least two populations of liposome-bound parvalbumin one of which has higher affinity to the liposomes (effective binding constant about 10(6) M-1) than the other one. The interaction is modulated by Ca2+ and Mg2+ ions and induces changes in properties of both parvalbumin and liposomes.

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.

Two partially unfolded states of Torpedo californica acetylcholinesterase.

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

Irreversible thermal denaturation of Torpedo californica acetylcholinesterase.

Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.

Efficacy and compliance with noninvasive positive pressure ventilation in patients with chronic respiratory failure.

STUDY OBJECTIVES: Previous studies have shown the acute effects of noninvasive positive pressure ventilation (NPPV) in chronic respiratory failure; however, information on the chronic effects of NPPV is limited. We examined the acute and chronic effects of NPPV on gas exchange, functional status, and respiratory mechanics in patients with chronic respiratory failure related to restrictive ventilatory disorders or COPD. DESIGN: Descriptive analysis of prospectively collected clinical data. SETTING: Inpatient noninvasive respiratory care unit and outpatient clinic of university hospital. PATIENTS: Forty patients with chronic respiratory failure (20 with severe COPD and 20 with restrictive ventilatory disorders). INTERVENTIONS AND MEASUREMENTS: All patients were admitted to a noninvasive respiratory care unit for 20 +/- 3 days for inpatient evaluation consisting of medical treatment, rehabilitation, and NPPV evaluation and instruction. NPPV was titrated via a ventilatory support system (BiPAP; Respironics Inc; Monroeville, PA) or a portable volume ventilator (PLV 102; Lifecare, Inc; Boulder, CO) to achieve a > or = 20% increase in baseline minute ventilation while monitoring gas exchange, expired volume, and clinical evidence of a decrease in the patient's work of breathing. RESULTS: The patients' mean age (+/- SD) was 65 +/- 9.7 years, and there was a 3:1 female:male predominance. In the noninvasive respiratory care unit, 36 patients used NPPV for 7.31 +/- 0.26 h/night. Four patients (three with COPD, one with restrictive disorder) withdrew from the study during the 3-week inpatient stay because they could not tolerate NPPV. Six patients (5 with COPD, 1 with restrictive disorder) used a portable volume ventilator and 34 patients used BiPAP (15 with COPD, 19 with restrictive disorders). At discharge, compared with at admission, daytime PaO2/fraction of inspired oxygen (FIO2) increased (327 +/- 10 vs 283 +/- 13 mm Hg; p = 0.01), PaCO2 was reduced (52 +/- 2 vs 67 +/- 3 mm Hg; p = 0.0001), and functional score increased (4.76 +/- 1.16 vs 2.7 +/- 1.64 arbitrary units (AUs); p < 0.01). Six months after discharge, improvements in PaO2/FIO2 (317 +/- 10 vs 283 +/- 13; p = 0.05), PaCO2 (52 +/- 2 vs 67 +/- 3 mm Hg; p = 0.0001), and functional score (5.66 +/- 0.41 vs 2.7 +/- 0.3 AUs; p < 0.001) were maintained compared with admission values. FVC, FEV1, and maximum inspired and expired mouth pressures were unchanged before and after long-term NPPV. Ten patients (7 with COPD, 3 with restrictive disorders) discontinued NPPV at 6 months, and 3 progressed to tracheostomy. The remaining 26 patients continued to use NPPV at the 6-month follow-up. They claimed to use NPPV for 7.23 +/- 0.24 h/night, but logged metered use was 4.5 +/- 0.58 h/night. Problems that required adjustment in either the mask (36%) or ventilator source (36%) included mask leaks (43%), skin irritation (22%), rhinitis (13%), aerophagia (13%), and discomfort from mask headgear (7%). CONCLUSION: NPPV acutely and chronically improves gas exchange and functional status in patients with chronic respiratory failure, but a significant number of patients do not tolerate NPPV on a chronic basis. Comprehensive follow-up is required to correct problems with NPPV and ensure optimal patient compliance.

Financial implications of noninvasive positive pressure ventilation (NPPV).

Noninvasive positive pressure ventilation (NPPV) is effective in the treatment of acute and chronic respiratory failure. However, the costs and financial balance between costs and diagnosis-related group (DRG) reimbursement for patients with moderate to severe respiratory failure treated with NPPV are unknown. We examined the costs and DRG reimbursement for 27 patients receiving Medicare referred with moderately severe respiratory failure for NPPV to the ventilator rehabilitation unit (VRU) at Temple University Hospital. This unit is one of four Health Care Financing Administration chronic ventilator-dependent demonstration sites that evaluates patients for NPPV, instructs them in home NPPV use, emphasizes rehabilitation, and uses strict cost accounting methods. Nineteen patients were treated with NPPV in the ICU and then referred to the VRU, and 8 patients were directly admitted for NPPV to the VRU. Patients were (mean +/- SE) 69 +/- 9 years age, 14 had severe COPD, and 13 had various restrictive disorders. All were hypercapneic at the time of hospital admission (restrictive 60 +/- 15; obstructive 67 +/- 3 mm Hg, PaCO2) with impaired lung mechanics and limited functional status. Patients averaged 8 +/- 15 days in the ICU, or 8 +/- 4.7 days on the medical floor prior to VRU transfer. The VRU length of stay averaged 20 +/- 18 days, for a total length of stay of 29 +/- 21 days. After implementation of NPPV, all patients had an improvement in gas exchange while spontaneously breathing and functional status that was maintained in follow-up. At 1 and 2 years of follow-up, 74% and 63% of patients were alive, respectively. Eleven patients were admitted with DRG 475 (respiratory system diagnosis with ventilator support); however, 16 of 27 patients were admitted across five different non-475 DRG codes with reimbursement rates ranging from $2,673 to $4,215. After DRG and outlier reimbursement, a total deficit of $261,948 remained (average deficit $9,701 per patient). However, individual patient deficits ranged from $1,113 to $32,892. Eighty-two percent of patients treated with NPPV incurred substantial financial losses that were underreimbursed across all assigned DRGs, including DRG 475, the highest-weighted DRG. We conclude that patients with moderate to severe respiratory failure receiving NPPV demonstrate an improvement in functional status and gas exchange that is maintained in follow-up. In addition, patients treated with NPPV incur high costs that are currently underreimbursed by the present DRG system. Newer DRG payment scales that recognize NPPV as specific treatment should be implemented.

Efficacy of a new full face mask for noninvasive positive pressure ventilation.

Previous studies have shown that noninvasive positive pressure ventilation (NPPV) improves gas exchange in acute and chronic respiratory failure. However, some patients are unable to tolerate NPPV due to air leaks around the mask, facial discomfort, and claustrophobia. A new mask that covers the entire face (Total, Respironics, Monroeville, Pa), attempts to overcome these obstacles. We studied the efficacy of NPPV via the Total face mask (TFM) in nine patients with chronic respiratory failure. In three patients, respiratory failure was due to chronic obstructive lung disease, and in six patients, it was secondary to restrictive disorders. None of the patients were previously able to tolerate NPPV via nasal (N) or nasal-oral (NO) masks. At baseline, all patients had impaired gas exchange with low PaO2/FIO2 (241 +/- 14), elevated PaCO2 (79 +/- 5 mm Hg), and poor functional status (1.89 +/- 1.45, on a scale of 1 to 7). After NPPV in the hospital for 7.1 +/- 1.5 h per night for 22 +/- 26 days, the PaCO2 fell to 59 +/- 3 mm Hg, and the PaO2/FIO2 rose to 304 +/- 27. Following nocturnal NPPV via the TFM for 6.7 +/- 1.5 h a night 6 +/- 5 weeks after hospital discharge, sustained improvements in PaCO2 (58 +/- 3 mm Hg, p < 0.05), PaO2/FIO2 (304 +/- 18), and functional status (5.38 +/- 1.06, p < 0.05) were observed. In four patients, measurements of respiratory rate, tidal volume, minute ventilation, dyspnea, discomfort with the face mask, and mask and mouth leaks were made during 30-min sessions of NPPV applied at constant levels via all three masks (N, NO, TFM). Discomfort with the face mask (0.38 +/- 0.18 vs 1.44 +/- 0.34 vs 2.38 +/- 0.32, p < 0.05) and mask leaks (0.44 +/- 0.18 vs 1.89 +/- 0.39 vs 1.89 +/- 0.35, p < 0.05) were least during NPPV via TFM compared with the N or NO masks, respectively. Moreover, expired tidal volume was highest (804 +/- 10 vs 498 +/- 9 vs 537 +/- 13 ml, p < 0.05) and PaCO2 lowest (51 +/- 2 vs 57 +/- 2 vs 58 +/- 3, p < 0.05) during NPPV via the TFM compared with N or NO masks. We conclude that NPPV delivered via a Total mask ensures a comfortable, stable patient-mask interface and improves gas exchange in selected patients intolerant of more conventional N or NO masks.

Overview of improving tolerance of long-term mechanical ventilation.

In summary, advances in critical care and mechanical ventilation have resulted in an increased salvage of critically ill patients, a number of whom require long-term ventilation as a means of life support. In addition, the total number of patients receiving long-term ventilation has increased dramatically over the past couple of years, and they now are treated in many different locations throughout the health care delivery system (the intensive care unit, general medical floor, specialized weaning units, and the home). Moreover, patients who require long-term ventilation usually suffer from a complex blend of medical, physiologic, and psychological disorders that may impair tolerance of chronic ventilation. Careful evaluation and systematic treatment of psychological, medical, and physiologic disorders, coupled with optimized ventilator settings, may improve patients' tolerance and maximize their functional capacity.

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions.

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.

Interactions of calcium binding proteins, parvalbumin and alpha-lactalbumin, with dipalmitoylphosphatidylcholine vesicles.

Interactions of Ca2+ binding proteins, pike (Esox lucius) parvalbumins pI 4.2 and 5.0, and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles were studied by means of scanning microcalorimetry and intrinsic tyrosine and tryptophan fluorescence methods. The interactions of pike parvalbumins are modulated by Ca2+ and Mg2+ binding to the protein and induce some changes in the physical properties of both the proteins and liposomes. Liposomes increased thermal stability of Ca2+-loaded parvalbumin and decreased thermal stability of both Mg2+-loaded and metal-free protein. The interaction of parvalbumin with liposomes affects the phase transition from gel to liquid-crystalline state in liposomes. Ca2+-loaded alpha-lactalbumin interacts with liposomes in its native state while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The results of the microcalorimetric and spectrofluorometric studies are supported by data obtained by means of gel-chromatography on Sepharose 4B. It may be suggested that these metal-modulated interactions of Ca2+-binding proteins with membranes have some functional significance.

[The effect of calcium and magnesium ions on the interaction of calcium-binding proteins parvalbumin and alpha-lactalbumin with dipalmitoylphosphatidylcholine vesicles]. / Vliianie ionov kal'tsiia i magniia na vzaimodeistvie kal'tsiisviazyvaiushchikh belkov parval'bumina i alpha-laktal'bumina s vezikulami dipal'mitoilfosfatidilkholina.

Interactions of the calcium binding proteins, like parvalbumins pI 4.2 and p15.0 and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles have been studied by means of scanning microcalorimetry and intrinsic tryptophan, tyrosine and phenylalanine fluorescence. The interactions are modulated by the Ca2+ and Mg2+ binding to the proteins and induce some changes in the physical properties of both the proteins and the liposomes. The liposomes increase the thermal stability of the Mg2+-loaded and metal-free parvalbumin. Ca2+-loaded alpha-lactalbumin interacts with the liposomes in its native state, while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The interactions of both proteins with the liposomes affect the phase transition from gel to liquid-crystalline state in the liposomes. The results of the microcalorimetric and spectrofluorometric studies are corroborated by the data obtained by means of gel-chromatography on Sepharose 4B.

[Biogenic monoamines of the brain and spinal cord pia mater in vertebrates]. / Biogennye monoaminy miagkoi obolochki golovnogo i spinnogo mozga pozvonochnykh.

Fluorimetric studies have been made on the content of adrenalin, noradrenaline, serotonin, dopamine, and tryptamine in the pial matter of the brain and spinal cord of fishes, birds and mammals including man. Using histochemical method with glyoxylic acid, biogenic monoamines were revealed in the adrenergic nerve fibers and monoaminocytes. Their total content in the pial matter of the brain is approximately the same in all vertebrates, being significantly lower in man. Higher concentration of adrenergic axons and lower amount of monoaminocytes in human subjects reveal the key role of the nervous influences in regulation of hemodynamics of the brain.

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin.

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca2+ in cells ([Ca2+]i). Ca2+ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984). The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca(2+)- and calmodulin-dependent protein phosphatase, Ca(2+)-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca(2+)- and calmodulin control of sperm motility in sea urchins.

Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer.

Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a "molten globule" upon mild thermal denaturation or upon chemical modification of a single non-conserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.

Oxidative stress transforms acetylcholinesterase to a molten-globule-like state.

Exposure of purified acetylcholinesterase from Torpedo california to a system generating oxygen radicals (viz. ascorbic acid/Fe(EDTA)2/H2O2) inactivated the enzyme. The enzyme retained its native dimeric form, but electrophoresis under denaturing conditions showed some cleavage of peptide bonds. Spectroscopic characterization revealed a shift to the red in the intrinsic fluorescence emission peak, a large decrease in molar ellipticity in the near UV with a much smaller decrease in the far UV, and increased binding of the amphiphilic probe, 1-anilino-8-naphthalene sulfonate, all relative to native enzyme. The treated enzyme was also highly susceptible to proteolysis. These data show that oxygen radical treatment converts acetylcholinesterase to a partially unfolded state, which retains most of its secondary structure but lacks substantial tertiary structure, thus resembling a 'molten globule' state. This model system may offer a mechanism for explaining the consequences of oxidative stress in vivo: partially unfolded proteins generated by oxidative stress may interact with molecular chaperons of the heat shock family, thus activating the heat-shock factor and, thereby, activating heat-shock genes.

Quality of life in survivors of prolonged mechanical ventilatory support.

OBJECTIVE: To examine the long-term quality of life (QOL) in a group of patients after prolonged mechanical ventilatory support. DESIGN: Prospective cohort study. SETTING: Outpatient follow-up. PATIENTS: Survivors of prolonged mechanical ventilatory support who were discharged from a ventilator rehabilitation unit (VRU). INTERVENTIONS: Measurement of health-related QOL using the Sickness Impact Profile (SIP). MEASUREMENTS AND MAIN RESULTS: Forty-six patients were contacted approximately 2 yrs after their discharge from the VRU and asked to complete the SIP. Twenty-five patients (age, 59 +/- 17 yrs; duration of mechanical ventilatory support, 45 +/- 36 days [mean +/- sd]) agreed to participate in this study and completed the SIP questionnaire 23 +/- 18 months after their discharge from the VRU. Patients' VRU stay was 29 +/- 21 days. Two patients were discharged with nocturnal ventilatory support, and the rest were completely weaned of mechanical ventilatory support before discharge. Fifteen patients (60%) were discharged to home, eight patients (32%) were discharged to a rehabilitation facility, and two patients (8%) were discharged to a skilled-care facility. Most patients had mild dysfunction, and the global SIP score was 12 +/- 10, the physical dimension score was 12 +/- 12, and the psychosocial dimension score was 9 +/- 11 (SIP scores range from 0 to 100, with higher scores indicating worse QOL). Subgroup analysis showed that postoperative patients had lower SIP scores compared with patients with chronic respiratory diseases (global SIP, 7 +/- 6 vs. 19 +/- 8; p <.05). Moreover, the patients in the postoperative group were older, but had similar SIP scores as patients who had acute lung injury (17 +/- 15). Global SIP scores correlated with age (r = -.40; p =.046), but not with duration of mechanical ventilatory support (r = -.23) or VRU admission Acute Physiology and Chronic Health Evaluation II scores (r = -.39; p =.06). CONCLUSIONS: In survivors of prolonged mechanical ventilatory support, using specific selection criteria shows that there is minimal impairment in the QOL at long-term follow-up. Although some patients continue to have moderate to severe limitations, it is the cause of respiratory failure and the underlying disease, rather than duration of ventilatory support, that have a significant impact on QOL.

A "molten globule" of Torpedo acetylcholinesterase undergoes thiol-disulfide exchange.

Torpedo acetylcholinesterase is a disulfide-linked homodimer containing three intramolecular disulfide bonds, as well as a single free thiol on Cys-231. We report that in a "molten globule" state, produced by 1.5 M guanidine hydrochloride, this enzyme undergoes rapid intramolecular thiol-disulfide exchange, in the absence of reducing agents, resulting in the production of novel species. Most strikingly, this results in appearance of enzyme monomers. Chemical modification of the free thiol group prevents these changes. Unfolded acetylcholinesterase, namely in 5 M guanidine hydrochloride, also undergoes intramolecular thiol-disulfide exchange, including production of enzyme monomers, but at a much lower rate. Our data show that the molten globule state, in contrast to the native and unfolded states, is both compact and flexible, thus being especially amenable to thiol-disulfide exchange.

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

Cys-231 of Torpedo californica acetylcholinesterase (EC 3.1.1.7) was selectively labeled with the mercury derivative of a stable nitroxyl radical. In 1.5 M guanidinium chloride, this conjugate exists in a molten globule state (MG), whereas in 5 M denaturant, it is in an unfolded state (U). The transition between the two states is reversible. In the MG, the label is highly immobilized, whereas in the U, it is almost freely rotating. The clearly distinct electron paramagnetic resonance (EPR) spectra of the two states permits the study of this transition. Upon elevating the guanidinium chloride concentration, a decrease in the EPR signal of the MG occurs concomitantly with an increase in the U signal, the total intensity of the EPR spectra remaining constant. This behavior is characteristic of a two-state transition. The thermodynamic characteristics of this transition (delta G0 and m), whether estimated directly from the EPR data or from both CD and fluorescence data analyzed by assuming a two-state scheme, are in good agreement.

Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.

The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.