Backbone dynamics of bacteriorhodopsin as studied by <Superscript>13</Superscript>C solid-state NMR spectroscopy

The surface dynamics of bacteriorhodopsin was examined by measurements of site-specific ¹³C–¹H dipolar couplings in [3-¹³C]Ala-labeled bacteriorhodopsin. Motions of slow or intermediate frequency (correlation time <50 µs) scale down ¹³C–¹H dipolar couplings according to the motional amplitude. The two-dimensional dipolar and chemical shift (DIPSHIFT) correlation technique was utilized to obtain the dipolar coupling strength for each resolved peak in the ¹³C MAS solid-state NMR spectrum, providing the molecular order parameter of the respective site. In addition to the rotation of the Ala methyl group, which scales the dipolar coupling to 1/3 of the rigid limit value, fluctuations of the C–C vector result in additional motional averaging. Typical order parameters measured for mobile sites in bacteriorhodopsin are between 0.25 and 0.29. These can be assigned to Ala103 of the C–D loop and Ala235 at the C-terminal -helix protruded from the membrane surface, and Ala196 of the F–G loop, as well as to Ala228 and Ala233 of the C-terminal -helix and Ala51 from the transmembrane -helix. Such order parameters departing significantly from the value of 0.33 for rotating methyl groups are obviously direct evidence for the presence of fluctuation motions of the Ala C–C vectors of intact preparations of fully hydrated, wild-type bacteriorhodopsin at ambient temperature. The order parameter for Ala160 from the expectantly more flexible E–F loop, however, is unavailable under highest-field NMR conditions, probably because increased chemical shift anisotropy together with intrinsic fluctuation motions result in an unresolved ¹³C NMR signal.     

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.     

Conformational changes of bacteriorhodopsin along the proton-conduction chain as studied with (13)C NMR of [3-(13)C]Ala-labeled protein: arg(82) may function as an information mediator.

We have recorded (13)C NMR spectra of [3-(13)C]Ala-labeled wild-type bacteriorhodopsin (bR) and its mutants at Arg(82), Asp(85), Glu(194), and Glu(204) along the extracellular proton transfer chain. The upfield and downfield displacements of the single carbon signals of Ala(196) (in the F-G loop) and Ala(126) (at the extracellular end of helix D), respectively, revealed conformational differences in E194D, E194Q, and E204Q from the wild type. The same kind of conformational change at Ala(126) was noted also in the Y83F mutant, which lacks the van der Waals contact between Tyr(83) and Ala(126) present in the wild type. The absence of a negative charge at Asp(85) in the site-directed mutant D85N induced global conformational changes, as manifested in displacements or suppression of peaks from the transmembrane helices, cytoplasmic loops, etc., as well as the local changes at Ala(126) and Ala(196) seen in the other mutants. Unexpectedly, no conformational change at Ala(126) was observed in R82Q (even though Asp(85) is protonated at pH 6) or in D85N/R82Q. The changes induced in the Ala(126) signal when Asp(85) is uncharged could be interpreted therefore in terms of displacement of the positive charge of Arg(82) toward Tyr(83), where Ala(126) is located. It is possible that disruption of the proton transfer chain after protonation of Asp(85) in the photocycle could cause the same kind of conformational change we detect at Ala(196) and Ala(126). If so, the latter change would be also the result of rearrangement of the side chain of Arg(82).     

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.

We have examined the (13)C-NMR spectra of [3-(13)C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mM NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) (13)C signals from the C-terminal alpha helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal alpha helix is deleted or site-directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.     

Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp 85 and deprotonation of Schiff base as studied by 13C NMR

According to previous X-ray diffraction studies, the D85N mutant of bacteriorhodopsin (bR) with unprotonated Schiff base assumes a protein conformation similar to that in the M photointermediate. We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N and D85N/D96N mutants at ambient temperature to examine how conformation and dynamics of the protein backbone are altered when the Schiff base is protonated (at pH 7) and unprotonated (at pH 10). Most notably, we found that the peak intensities of three to four [3-(13)C]Ala-labeled residues from the transmembrane alpha-helices, including Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D85N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-)(5) or 10(-)(4) s, which interferes with proton decoupling frequency or frequency of magic angle spinning, respectively, essential for an attempted peak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of transmembrane helices upon deprotonation of Schiff base and the formation of the M-like state in the absence of illumination. The spectra detected more rapid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-)(4) to 10(-)(5) s. Conformational changes in the transmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-(13)C-labeled Val 49 (helix B), 69 (B-C loop), and [3-(13)C]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side substantially modulated the conformation and dynamics of the extracellular region through long-distance interaction.     

Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein

The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix. It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence. With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues. We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants. Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport. The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues. This extension superimposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus. This is similar to the helix D and F regions of the yeast PTP. Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D. All other transport-inhibiting mutations are located within the TM helices. Mutations that yield LIEs of <6% are all, except for four, within helices. The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D. The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6%. Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D. Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity. A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies.     

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10(5) Hz in view of the suppressed peaks from [3-(13)C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal alpha-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignment of full-length human uracil DNA glycosylase UNG2

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.     

Quantitative analysis of helix-coil transition of block copolypeptide, Glu12-Ala12, by combined use of CD and NMR spectroscopy

To investigate helix-coil transition mechanisms, conformations of Glu12-Ala12, EA, in aqueous solution have been studied in detail over the pH range from 2 to 8 and the temperature range from 20 to 60 degrees C using CD and NMR spectroscopy. The 750-MHz NMR spectra displayed excellent dispersion of the backbone amide proton signals, and permitted essentially complete sequence-specific resonance assignments. These assignments, together with short- and medium-range nuclear Overhauser effect (NOE) constraints and coupling constants, enable us to analyze conformational characteristics of all the residues in the EA peptide individually. A combined use of CD and NMR techniques reveals that the EA peptide assumes a stable alpha-helix from Glu12 to Ala19 in 0.1 M NaCl solution at 20 degrees C above pH 7. The alpha-helix is getting longer as decreasing pH. Below pH 4, the peptide assumes the longest alpha-helix from Glu3 to Ala23. The important observation of the present study is that the helix-coil transition occurs stepwise, residue by residue, from both the N- and C-termini of the alpha-helix. No conformational equilibrium between the helical and random-coil states is detected for the residues in the central region of the alpha-helix. Quantitative analysis of temperature-induced helix-to-coil transitions at various pHs provides a pH-independent residual enthalpy change delta H(r) = 0.95 kcal res(-1). Similar values have been reported for a 50-residue alanine-rich peptide (1.2 kcal res(-1)), poly-L-glutamate (1.1 kcal res(-1)), poly-L-lysine (1.1 kcal res(-1)), and poly-L-alanine (0.86 kcal res(-1)). Those investigations, along with our present result, suggest that delta H(r) is mainly determined by the transformation of the backbone associated with the disruption of the intramolecular hydrogen bond. These results should increase our understanding of the helix-coil transition.     

Changing the Metal Binding Specificity of Superoxide Dismutase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB-27 by a Single Mutation

Metal binding of superoxide dismutase from Thermus thermophilus HB27 was analyzed by comparing the related structures and sequences from different origins. Mutants (Ile166Leu, Asp167Glu, and Ile166Leu-Asp167Glu) were prepared and characterized. The mutants Asp167Glu and Ile166Leu-Asp167Glu changed their binding specificities from manganese to iron, which were manifested by the differences in color of the enzyme solutions and by flame atomic absorption analysis. Specific activities of the three mutants were 112, 52, and 62% of that of the wild-type enzyme, respectively. Asp167Glu and Ile166Leu-Asp167Glu only retained 6.8 and 6.1%, respectively, of the original activities after dialysis against 1 mM EDTA. Tryptophan fluorescence measurement and native gel electrophoresis implied that the three mutants could fold into a less condensed structure. Their folding and changes in the ion binding sites of the modeled structures might be the reason for their low affinities to metal ions. These findings increased our understanding of metal binding specificity of superoxide dismutase.     

Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products

NADPH-dependent 2,4-dienoyl-CoA reductase (DCR) is one of the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids. Mutants of Escherichia coli DCR were generated by site-directed mutagenesis to explore the molecular mechanism of this enzyme. The Tyr166Phe mutant, which was expected to be inactive due to the loss of its putative proton donor residue, exhibited 27% of the wild-type activity. However, the product of the reduction was 3-enoyl-CoA instead of 2-enoyl-CoA, the normal product. Glu164 seems to function as proton donor in the Tyr166Phe mutant, because the Tyr166Phe/ Glu164Gln double mutant was inactive whereas the Glu164Ala mutant exhibited low but significant activity. His252 is important for the efficient operation of Tyr166 because a His252Ala mutation by itself reduced the activity of DCR by 3 orders of magnitude, whereas the Tyr166Phe/His252Ala double mutation exhibited 4.4% of the wild-type activity. This data supports a mechanism that has Tyr166 with the assistance of His252 acting as proton donor in the wild-type enzyme to produce 2-enoyl-CoA, whereas Glu164 serves as the proton donor in the absence of Tyr166 to yield 3-enoyl-CoA. A Cys337Ala mutation, which resulted in the loss of most of the iron and acid-labile sulfur, decreased the reductase activity more than 1000-fold. This observation agrees with the proposed operation of an intramolecular electron transport chain that is essential for the effective catalysis of E. coli DCR.     

Evidence for long range allosteric interactions between the extracellular and cytoplasmic parts of bacteriorhodopsin from the mutant R82A and its second site revertant R82A/G231C

Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/G231C. (i) In the double mutants R82A/G72C and R82A/A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay (tau approximately 8 ms), in R82A it occurs already during the first phase of the M-rise (tau < 1 microseconds). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical G-protein-coupled receptors.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

Selection of <Emphasis Type="Italic">Escherichia coli</Emphasis> Glutamate Decarboxylase Active at Neutral pH from a Focused Library

Bacterial glutamate decarboxylase (GAD) converts glutamate (Glu) into γ-aminobutyric acid (GABA) at acidic conditions. Since the reaction consumes a proton per GABA synthesis, cells use this reaction to survive in the acidic environments. Characteristically, the enzyme displays a sigmoidal decrease in its activity as pH rises becoming completely inactive at or above pH 6. This cooperative activity loss is accompanied by several distinct structural changes. Previously, by examining structures at acidic and neutral pH, two key regions had been chosen and mutated to break the cooperativity; Glu89 and C-terminal 15 residues. In this study, we included Asp86 in candidate key residues for mutation to break the cooperativity of GAD. We devised a selection strategy according to which only Escherichia coli cells expressing a variant GAD that was active at neutral pH could survive. In this scheme, an alanine (Ala) auxotroph was rescued by the intracellular synthesis of GABA that was subsequently converted into Ala by heterologously expressed GABA-pyruvate transaminase. New GAD variants were readily selected using this strategy and the most of them indeed had a mutation at residue 86. The results suggest that the role of Asp86 in the wild-type enzyme might be the same as Glu89; to make GAD keep its activity only at acidic environments. Characterization of representative variants are also presented.     

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.