Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

A normal mode analysis of structural plasticity in the biomolecular motor F(1)-ATPase

Normal modes have been used to explore the inherent flexibility of the alpha, beta and gamma subunits of F(1)-ATPase in isolation and as part of the alpha(3)beta(3)gamma complex. It was found that the structural plasticity of the gamma and beta subunits, in particular, correlates with their functions. The N and C-terminal helices forming the coiled-coil domain of the gamma subunit are highly flexible in the isolated subunit, but more rigid in the alpha(3)beta(3)gamma complex due to interactions with other subunits. The globular domain of the gamma subunit is structurally relatively rigid when isolated and in the alpha(3)beta(3)gamma complex; this is important for its functional role in coupling the F(0) and F(1) complex of ATP synthase and in inducing the conformational changes of the beta subunits in synthesis. Most important, the character of the lowest-frequency modes of the beta(E) subunit is highly correlated with the large beta(E) --> beta(TP) transition. This holds for the C-terminal domain and the nucleotide-binding domain, which undergo significant conformational transitions in the functional cycle of F(1)-ATPase. This is most evident in the ligand-free beta(E) subunit; the flexibility in the nucleotide-binding domain is reduced somewhat in the beta(TP) subunit in the presence of Mg(2+).ATP. The low-frequency modes of the alpha(3)beta(3)gamma complex show that the motions of the globular domain of the gamma subunit and of the C-terminal and nucleotide binding domains of the beta(E) subunits are coupled, in accord with their function. Overall, the normal mode analysis reveals that F(1)-ATPase, like other macromolecular assemblies, has the intrinsic structural flexibility required for its function encoded in its sequence and three-dimensional structure. This inherent plasticity is an essential aspect of assuring a small free energy cost for the large-scale conformational transition that occurs in molecular motors.     

pH dependence of the flash-induced S-state transitions in the oxygen-evolving center of photosystem II from Thermosynechoccocus elongatus as revealed by Fourier transform infrared spectroscopy

pH dependence of the efficiencies of the flash-induced S-state transitions in the oxygen-evolving center (OEC) was studied by means of Fourier transform infrared (FTIR) difference spectroscopy using photosystem II (PSII) core complexes from the thermophilic cyanobacterium Thermosynechoccocus elongatus. The PSII core complexes dark-adapted at different pHs in the presence of ferricyanide as an electron acceptor were excited by four consecutive saturating laser flashes, and FTIR difference spectra induced by each flash were recorded in the region of 1800-1200 cm(-1). Each difference spectrum was fitted with a linear combination of standard spectra measured at pH 6.0, which represent the spectra upon individual S-state transitions, and the transition efficiencies were estimated from the fitting parameters. It was found that the S1 --> S2 transition probability is independent of pH throughout the pH region of 3.5-9.5, while the S2 --> S3, S3 --> S0, and S0 --> S1 transition probabilities decrease at acidic pH with pK values of 3.6 +/- 0.2, 4.2 +/- 0.3, and 4.7 +/- 0.5, respectively. These findings, i.e., the pH-independent S1 --> S2 transition probability and the pK values for the inhibition in the acidic range of the other three transitions, were in good agreement with recent results obtained by electron paramagnetic resonance measurements for PSII-enriched membranes of spinach [Bernát, G., Morvaridi, F., Feyziyev, Y., and Styring, S. (2002) Biochemistry 41, 5830-5843]. On the basis of this correspondence for quite different types of PSII preparations exhibiting marked difference in the pH dependence of the apparent proton release pattern, it is concluded that the inhibition of the S2 --> S3, S3 --> S0, and S0 --> S1 transitions in the acidic region is an inherent property of the OEC. This feature probably reflects proton release from substrate water in these three transitions. On the other hand, all of the S-state transitions remained generally efficient up to pH 9.5 in the alkaline region, except for a slight decrease of the S3 --> S0 transition probability above pH 8 (pK approximately 10). This observation partly differs from the tendency reported for spinach preparations, suggesting that a mechanism different from that in the acidic region is responsible for the transition efficiencies in the alkaline region.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

The thermotropic phase behaviour and phase structure of a homologous series of racemic beta-D-galactosyl dialkylglycerols studied by differential scanning calorimetry and X-ray diffraction

The thermotropic phase behaviour of aqueous dispersions of some synthetic 1,2-di-O-alkyl-3-O-(beta-D-galactosyl)-rac-glycerols (rac-beta-D-GalDAGs) with both odd and even hydrocarbon chain lengths was studied by differential scanning calorimetry (DSC), small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction. DSC heating curves show a complex pattern of lamellar (L) and nonlamellar (NL) phase polymorphism dependent on the sample's thermal history. On cooling from 95 degrees C and immediate reheating, rac-beta-D-GalDAGs typically show a single, strongly energetic phase transition, corresponding to either a lamellar gel/liquid-crystalline (L(beta)/L(alpha)) phase transition (N< or =15 carbon atoms) or a lamellar gel/inverted hexagonal (L(beta)/H(II)) phase transition (N> or =16). At higher temperatures, some shorter chain compounds (N=10-13) exhibit additional endothermic phase transitions, identified as L/NL phase transitions using SAXS/WAXS. The NL morphology and the number of associated intermediate transitions vary with hydrocarbon chain length. Typically, at temperatures just above the L(alpha) phase boundary, a region of phase coexistence consisting of two inverted cubic (Q(II)) phases are observed. The space group of the cubic phase seen on initial heating has not been determined; however, on further heating, this Q(II) phase disappears, enabling the identification of the second Q(II) phase as Pn3 m (space group Q(224)). Only the Pn3 m phase is seen on cooling. Under suitable annealing conditions, rac-beta-D-GalDAGs rapidly form highly ordered lamellar-crystalline (L(c)) phases at temperatures above (N< or =15) or below (N=16-18) the L(beta)/L(alpha) phase transition temperature (T(m)). In the N< or =15 chain length lipids, DSC heating curves show two overlapping, highly energetic, endothermic peaks on heating above T(m); corresponding changes in the first-order spacings are observed by SAXS, accompanied by two different, complex patterns of reflections in the WAXS region. The WAXS data show that there is a difference in hydrocarbon chain packing, but no difference in bilayer dimensions or hydrocarbon chain tilt for these two L(c) phases (termed L(c1) and L(c2), respectively). Continued heating of suitably annealed, shorter chain rac-beta-D-GalDAGs from the L(c2) phase results in a phase transition to an L(alpha) phase and, on further heating, to the same Q(II) or H(II) phases observed on first heating. On reheating annealed samples with longer chain lengths, a subgel phase is formed. This is characterized by a single, poorly energetic endotherm visible below the T(m). SAXS/WAXS identifies this event as an L(c)/L(beta) phase transition. However, the WAXS reflections in the di-16:0 lipid do not entirely correspond to the reflections seen for either the L(c1) or L(c2) phases present in the shorter chain rac-beta-D-GalDAGs; rather these consist of a combination of L(c1), L(c2) and L(beta) reflections, consistent with DSC data where all three phase transitions occur within a span of 5 degrees C. At very long chain lengths (N> or =19), the L(beta)/L(c) conversion process is so slow that no L(c) phases are formed over the time scale of our experiments. The L(beta)/L(c) phase conversion process is significantly faster than that seen in the corresponding rac-beta-D-GlcDAGs, but is slower than in the 1,2-sn-beta-D-GalDAGs already studied. The L(alpha)/NL phase transition temperatures are also higher in the rac-beta-D-GalDAGs than in the corresponding rac-beta-D-GlcDAGs, suggesting that the orientation of the hydroxyl at position 4 and the chirality of the glycerol molecule in the lipid/water interface influence both the L(c) and NL phase properties of these lipids, probably by controlling the relative positions of hydrogen bond donors and acceptors in the polar region of the membrane.     

FTIR detection of water reactions in the oxygen-evolving centre of photosystem II

Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700-3500 cm(-1) region in FTIR difference spectra during S-state cycling. In the S1-->S2 transition, a band shift from 3588 to 3617 cm(-1) was observed, indicative of a weakened H-bond. Decoupling experiments using D2O:H2O (1:1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S2 formation. In the S2-->S3, S3-->S0 and S0-->S1 transitions, negative bands were observed at 3634, 3621 and 3612 cm(-1), respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600-1300 cm-(1)) as 'fingerprints' of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S2-->S3, S3-->S0 and S0-->S1 transitions was consistent with a view that protons are released in the three transitions other than S1-->S2, while relatively high susceptibility to dehydration in the S2-->S3 and S3-->S0 transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.     

Stability of a homo-dimeric Ca(2+)-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum.

Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca(2+)-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5-1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N2-->2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca(2+)-free and Ca(2+)-loaded protein were found to be 65 and 85 degrees C, the enthalpy changes delta Hcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and delta Hcal on the GdmCl concentration allow the molar heat capacity change delta Cp to be determined. As a result, delta Cp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy delta G degree calculated from delta Hcal and TM, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca(2+)-free or the Ca(2+)-loaded protein, respectively. Within the limits of error, the delta G degree values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results.     

Effect of myelin basic protein on the thermotropic behavior of aqueous dispersions of neutral and anionic glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine

The thermotropic behavior of the natural glycosphingolipids galactosylceramide, asialo-Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-Cer (GM1), sulfatide, GM1, NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer (GD1a), and NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc8-2 alpha NeuAc)beta 1-4Glc beta 1-1 Cer (GT1b), and their mixtures with dipalmitoylphosphatidylcholine (DPPC) in the presence of myelin basic protein (MBP) was studied by high sensitivity differential scanning calorimetry. The transition temperature of DPPC, galactosylceramide, and asialo-GM1 is affected little by MBP while their transition enthalpy is decreased in proportion to the amount of protein in the mixture. The thermotropic behavior of anionic glycosphingolipids is considerably perturbed by MBP. The transition temperature of gangliosides increases in the presence of MBP, whereas that of sulfatide decreases. The enthalpy of the transition of anionic glycosphingolipids increases markedly in the presence of MBP. The excess heat capacity function of these systems can be resolved into two independent phase transitions. Phase separation of enriched lipid/protein domains occurs in a magnitude that depends on the amount of MBP; the rest of the lipid phase exhibits some altered thermodynamic properties. In mixtures of glycosphingolipids with DPPC, phase separation is also present but no phase transition with the characteristic of pure DPPC is found. MBP is changing the properties of the lipid mixture as a whole and does not interact exclusively with the glycosphingolipids. The proportion of MBP required to produce the maximal changes is greater the greater the complexity of the glycosphingolipids polar head group. Relatively small variations of the amount of MBP induce large shifts in the proportion of the different phases present.     

Rhythms of high-grade block in an ionic model of a strand of regionally ischemic ventricular muscle

Electrical alternans, a beat-to-beat alternation in the electrocardiogram or electrogram, is frequently seen during the first few minutes of acute myocardial ischemia, and is often immediately followed by malignant cardiac arrhythmias such as ventricular tachycardia and ventricular fibrillation. As ischemia progresses, higher-order periodic rhythms (e.g., period-4) can replace the period-2 alternans rhythm. This is also seen in modelling work on a two-dimensional (2-D) sheet of regionally ischemic ventricular muscle. In addition, in the experimental work, ventricular arrhythmias are overwhelmingly seen only after the higher-order rhythms arise. We investigate an ionic model of a strand of ischemic ventricular muscle, constructed as a 3-cm-long 1-D cable with a centrally located 1-cm-long segment exposed to an elevated extracellular potassium concentration ([K(+)](o)). As [K(+)](o) is raised in this "ischemic segment" to represent one major effect of ongoing ischemia, the sequence of rhythms {1:1-->2:2 (alternans)-->2:1} is seen. With further increase in [K(+)](o), one sees higher-order periodic 2N:M rhythms {2:1-->4:2-->4:1-->6:2-->6:1-->8:2-->8:1}. In a 2N:M cycle, only M of the 2N action potentials generated at the proximal end of the cable successfully traverse the ischemic segment, with the remaining ones being blocked within the ischemic segment. Finally, there is a transition to complete block {8:1-->2:0-->1:0} (in an n:0 rhythm, all action potentials die out within the ischemic segment). Changing the length of the ischemic segment results in different rhythms and transitions being seen: e.g., when the ischemic segment is 2 cm long, the period-6 rhythms are not seen; when it is 0.5 cm long, there is a 3:1 rhythm interposed between the 2:1 and 1:0 rhythms. We discuss the relevance of our results to the experimental observations on the higher-order rhythms that presage reentrant ischemic ventricular arrhythmias.     

Phase behavior and glass transition of 1,2-dioleoylphosphatidylethanolamine (DOPE) dehydrated in the presence of sucrose

The effect of sucrose on the phase behavior of 1,2-dioleoylphosphatidylethanolamine (DOPE) as a function of hydration was studied using differential scanning calorimetry and X-ray diffraction. DOPE/sucrose/water dispersions were dehydrated at osmotic pressures (Pi) ranging from 2 to 300 MPa at 30 degrees C and 0 degrees C. The hexagonal II-to-lamellar gel (H(II)-->L(beta)) thermotropic phase transition was observed during cooling in mixtures dehydrated at Pior=57 MPa, the H(II)-->L(beta) thermotropic phase transition was precluded when sucrose entered the rigid glassy state while the lipid was in the H(II) phase. Sucrose also hindered the H(II)-to-lamellar crystalline (L(c)), and H(II)-to-inverted ribbon (P(delta)) lyotropic phase transitions, which occurred in pure DOPE. Although the L(c) phase was observed in dehydrated 2:1 (mole ratio) DOPE/sucrose mixtures, it did not form in mixtures with higher sucrose contents (1:1 and 1:2 mixtures). The impact of sucrose on formation of the ordered phases (i.e., the L(c), L(beta), and P(delta) phases) of DOPE was explained as a trapping of DOPE in a metastable H(II) phase due to increased viscosity of the sucrose matrix. In addition, a glass transition of DOPE in the H(II) phase was observed, which we believe is the first report of a glass transition in phospholipids.     

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.     

Computer analysis of sub-parameters of depurination and depolymerization rate constants in Feulgen DNA hydrolysis

Feulgen DNA hydrolysis curves derived from cytofluorometry at various temperatures and HCl concentrations were computer analyzed with least squares fit to Bateman function. By comparing the depurination (k1) and depolymerization (k2) rate constants at different hydrolysis conditions, it was found that the two parameters of temperature and HCl concentration can be expressed as k = AN2 X exp (-B/T), where A and B are constants, N is the HCl concentrations, and T is the absolute temperature. From the analysis of Feulgen hydrolysis curves with 2N HCl at various temperatures, it was calculated that A = 5.3590 X 10(14) and B = 12133.543, for k1, and A = 6.2401 X 10(14) and B = 12181.660, for k2 for mouse 4C hepatocytes fixed with absolute methanol. Computer generated theoretical hydrolysis curves using the above k1 and k2 values were compared with experimental curves at various temperatures and HCl-concentrations. The two types of hydrolysis curves coincided with each other when 1-3 N HCl was used at temperatures between 30-40 degrees C. The peak times of hydrolysis curves at different conditions determined by experimental analysis and theoretical estimations also coincided reasonably well with each other. The physico-chemical phenomena underlying the equation designating k1 and k2 values are discussed.     

The enigma of the liganded hemoglobin end state: a novel quaternary structure of human carbonmonoxy hemoglobin

The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

Synthesis of aminoethyl glycosides of the carbohydrate chains of glycolipids Gb3, Gb4 i Gb5

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl)-beta- D-glucopyranoside with ethyl 2,3,4,6-tetra-O-benzyl- and ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio-alpha-D-galactopyranoside in the presence of methyl trifluoromethanesulfonate led to trisaccharide 2-azidoethyl (2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-(1-->4)- (2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl)-(1-->4)2,3,6-tri-O- benzoyl-beta-D-glucopyranoside and its 3"-O-acetylated analogue, 2-azidoethyl (3-O-acetyl-2,4,6-tri-O-benzyl- alpha-D-galactopyranosyl)-(1-->4)-(2,3,6-tri-O-benzoyl-beta-D- galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside, in yields of 85 and 83%, respectively. Deacetylation of the latter compound and subsequent glycosylation with 4-trichloroacetamidophenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-trichloroacetamido-beta-D- galactopyranoside and 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D- galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in the corresponding selectively protected derivatives of tetrasaccharide GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc beta-OCH2CH2N3 and pentasaccharide Gal(beta 1-->3)GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc beta-OCH2CH2N3 in 88 and 73% yields, respectively. Removal of O-protecting groups, substitution of acetyl group for N-trichloroacetyl group, and reduction of the aglycone azide group resulted in the target 2-aminoethyl globo-tri-, -tetra-, and -pentasaccharide, respectively.     

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.     

No evidence from FTIR difference spectroscopy that aspartate-342 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Asp342 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn4 cluster. To determine if D1-Asp342 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions, the FTIR difference spectra of the individual S state transitions in D1-D342N mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that the mid-frequency (1800-1200 cm-1) FTIR difference spectra of wild-type and D1-D342N PSII particles are essentially identical. Importantly, the mutation alters none of the carboxylate vibrational modes that are present in the wild-type spectra. The absence of significant mutation-induced spectral alterations in D1-D342N PSII particles shows that the oxidation of the Mn4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp342 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. One explanation of these data is that D1-Asp342 ligates a Mn ion that does not increase its charge or oxidation state during any of these S state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Asp342 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn4 cluster during the S1 --> S2 transition be localized on the Mn ion that is ligated by the alpha-COO- group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn4 cluster during the S0 --> S1 and S2 --> S3 transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Asp342, or D1-Ala344. In separate experiments that were conducted with l-[1-13C]alanine, we found no evidence that D1-Asp342 ligates the same Mn ion that is ligated by the alpha-COO- group of D1-Ala344.     

Three-state protein folding: experimental determination of free-energy profile

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.