Impact of mutations within the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II D1 protein on the kinetics of photoactivation and H2O-oxidation in Synechocystis sp. PCC6803.

Mutations D1-D59N and D1-D61E in the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein [Chu, H. A., Nguyen, A. P., and Debus (1995), Biochemistry 34, 5839-5858] were further characterized in terms of S-state cycling and photoactivation. Bare platinum electrode measurements of centrifugally deposited O2-evolving membranes isolated from the a-b loop mutants demonstrated a retarded appearance of O2 following single turnover flashes, although not to the extent of retardation seen in the Deltapsb0 mutant, which lacks the extrinsic manganese-stabilizing protein (MSP). Double flash measurements indicate that retarded O2 release in mutants coincides with a decrease in overall PSII turnover during the S3-[S4]-S0 transition. S2 and S3 decay measurements in the isolated membranes indicate that D1-D59N and D1-D61E have faster decays of these higher S-states in contrast to slowed decays in the Deltapsb0 mutant. Measurements of the flash interval dependence of photoactivation indicate that intermediates of photoactivation [light-dependent assembly of the (Mn)4 complex] are highly destabilized in the a-b loop mutants compared to both DeltapsbO and the wild-type: flash intervals of greater than 2 s result in the nearly complete decay of unstable photointermediate(s) in the D1-D59N and D1-D61E samples, whereas a similar loss does not occur until intervals even greater than 10 s in the DeltapsbO and wild-type samples. These results are consistent with a role for the residues D1-D59 and D1-D61 in modulating the redox properties of the higher S-states and, also, possibly in the binding the calcium ion involved in photoactivation.     

Native N-Terminus Nitrophorin 2 from the Kissing Bug: Similarities to and Differences from NP2(D1A)

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R.?E. Berry, T.?K. Shokhireva, I. Filippov, M.?N. Shokhirev, H. Zhang, F.?A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.     

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.     

Axial ligand complexes of the<Emphasis Type="Italic"> Rhodnius</Emphasis> nitrophorins: reduction potentials,binding constants,EPR spectra,and structures of the 4-iodopyrazole and imidazole complexes of NP4

Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius prolixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G–H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the relative binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar.Abbreviations NP1–4 nitrophorins 1–4 from Rhodnius prolixus - 4IPzH 4-iodopyrazole - ImH imidazole - Hm histamine - NO nitric oxide - NOS nitric oxide synthase     

Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.     

The crystal structure of nitrophorin 2. A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.     

Safety and immunogenicity of heterologous boost immunization with an adenovirus type-5-vectored and protein-subunit-based COVID-19 vaccine (Convidecia/ZF2001): A randomized,observer-blinded,placebo-controlled trial

BackgroundHeterologous boost vaccination has been proposed as an option to elicit stronger and broader, or longer-lasting immunity. We assessed the safety and immunogenicity of heterologous immunization with a recombinant adenovirus type-5-vectored Coronavirus Disease 2019 (COVID-19) vaccine (Convidecia, hereafter referred to as CV) and a protein-subunit-based COVID-19 vaccine (ZF2001, hereafter referred to as ZF).Methods and findingsWe conducted a randomized, observer-blinded, placebo-controlled trial, in which healthy adults aged 18 years or older, who have received 1 dose of Convidecia, with no history of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, were recruited in Jiangsu, China. Sixty participants were randomly assigned (2:1) to receive either 1 dose of ZF2001 or placebo control (trivalent inactivated influenza vaccine (TIV)) administered at 28 days after priming, and received the third injection with ZF2001 at 5 months, referred to as CV/ZF/ZF (D0-D28-M5) and CV/ZF (D0-M5) regimen, respectively. Sixty participants were randomly assigned (2:1) to receive either 1 dose of ZF2001 or TIV administered at 56 days after priming, and received the third injection with ZF2001 at 6 months, referred to as CV/ZF/ZF (D0-D56-M6) and CV/ZF (D0-M6) regimen, respectively. Participants and investigators were masked to the vaccine received but not to the boosting interval. Primary endpoints were the geometric mean titer (GMT) of neutralizing antibodies against wild-type SARS-CoV-2 and 7-day solicited adverse reactions. The primary analysis was done in the intention-to-treat population. Between April 7, 2021 and May 6, 2021, 120 eligible participants were randomly assigned to receive ZF2001/ZF2001 (n = 40) or TIV/ZF2001 (n = 20) 28 days and 5 months post priming, and receive ZF2001/ZF2001 (n = 40) or TIV/ZF2001 (n = 20) 56 days and 6 months post priming. Of them, 7 participants did not receive the third injection with ZF2001. A total of 26 participants (21.7%) reported solicited adverse reactions within 7 days post boost vaccinations, and all the reported adverse reactions were mild, with 13 (32.5%) in CV/ZF/ZF (D0-D28-M5) regimen, 7 (35.0%) in CV/ZF (D0- M5) regimen, 4 (10.0%) in CV/ZF/ZF (D0-D56-M6) regimen, and 2 (10.0%) in CV/ZF (D0-M6) regimen, respectively. At 14 days post first boost, GMTs of neutralizing antibodies in recipients receiving ZF2001 at 28 days and 56 days post priming were 18.7 (95% CI 13.7 to 25.5) and 25.9 (17.0 to 39.3), respectively, with geometric mean ratios of 2.0 (1.2 to 3.5) and 3.4 (1.8 to 6.4) compared to TIV. GMTs at 14 days after second boost of neutralizing antibodies increased to 107.2 (73.7 to 155.8) in CV/ZF/ZF (D0-D28-M5) regimen and 141.2 (83.4 to 238.8) in CV/ZF/ZF (D0-D56-M6) regimen. Two-dose schedules of CV/ZF (D0-M5) and CV/ZF (D0-M6) induced antibody levels comparable with that elicited by 3-dose schedules, with GMTs of 90.5 (45.6, 179.8) and 94.1 (44.0, 200.9), respectively. Study limitations include the absence of vaccine effectiveness in a real-world setting and current lack of immune persistence data.ConclusionsHeterologous boosting with ZF2001 following primary vaccination with Convidecia is more immunogenic than a single dose of Convidecia and is not associated with safety concerns. These results support flexibility in cooperating viral vectored and recombinant protein vaccines.Trial registrationStudy on Heterologous Prime-boost of Recombinant COVID-19 Vaccine (Ad5 Vector) and RBD-based Protein Subunit Vaccine; ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT04833101","term_id":"NCT04833101"}}NCT04833101.

In a randomized controlled trial, Pengfei Jin and colleagues assess the safety and immunogenicity of heterologous immunization with a recombinant adenovirus type-5-vectored COVID-19 vaccine and a protein-subunit-based COVID-19 vaccine.     

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on ? His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe(II)-His bond breakage in heme proteins.     

Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus. Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3

We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism. Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane. In contrast to a previous report (Fee, J. A., Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000. Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1. A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.     

Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2

In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.     

Assignment of ferriheme resonances for high- and low-spin forms of nitrophorin 3 by H and C NMR spectroscopy and comparison to nitrophorin 2: Heme pocket structural similarities and differences

Nitrophorin 3 (NP3) is the only one of the four major NO-binding heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus (also called the Kissing Bug) for which it has not been possible to obtain crystals of diffraction quality for structure determination by X-ray crystallography. Thus we have used NMR spectroscopy, mainly of the hyperfine-shifted ferriheme substituent resonances, to learn about the similarities and differences in the heme pocket and the iron active site of NP3 as compared to NP2, which has previously been well-characterized by both X-ray crystallography and NMR spectroscopy. Only one residue in the heme pocket differs between the two, F27 of NP2 is Y27 for NP3; in both cases this residue is expected to interact strongly with the 2-vinyl side chain of the B heme rotational isomer or the 4-vinyl of the A heme rotational isomer. Both the high-spin (S = 5/2) aquo complex, NP3-H₂O, and the low-spin (S = 1/2) N-methylimidazole (NMeIm) complex of NP3 have been studied. It is found that the chemical shifts of the protons of both forms are similar to those of the corresponding NP2 complexes, but with minor differences that indicate a slightly different angle for the proximal histidine (H57) ligand plane. The B heme rotational isomer is preferred by both NP3 and NP2 in both spin states, but to a greater extent when phenylalanine is present at position 27 (A:B = 1:8 for NP2, 1:6 for NP3-Y27F, 1:4 for NP3, and 1:3 for NP2-F27Y). Careful analysis of the 5Me and 8Me shifts of the A and B isomers of the two high-spin nitrophorins leads to the conclusion that the heme environment for the two isomers differs in some way that cannot be explained at the present time. The kinetics of deprotonation of the aquo ligand of the high-spin complexes of NP2 and NP3 are very different, with NP2 giving well-resolved high-spin aquo and “low-spin” hydroxo proton NMR spectra until close to the end of the titration, while NP3 exhibits broadened ¹H NMR spectra indicative of an intermediate-rate of exchange on the NMR timescale between the two forms throughout the titration. The heme methyl shifts of NP2-OH are similar in magnitude and spread to those of NP2-CN, while those of metmyoglobin-hydroxo complexes are much larger in magnitude but not spread. It is concluded that the hydroxo complex of NP2 is likely S = 1/2 with a mixed(d_xy)²(d_xz, d_yz)³/(d_xy)¹(d_xz, d_yz)⁴ electron configuration, while those of metMb-OH are likely S = 1/2,3/2 mixed spin systems.     

Refolding, purification, and characterization of a loop deletion mutant of human Bcl-2 from bacterial inclusion bodies

This report describes the cloning of recombinant human Bcl-2, in which the putative disordered loop region has been replaced with a flexible linker and the hydrophobic C-terminus has been replaced with a 6xHis tag (Bcl-2(6-32)-AAAA-Bcl-2(86-206)-HHHHHH, abbreviation rhBcl-2; amino acid numbering excludes the initiating methionine). This protein was expressed in Escherichia coli where it accumulated in insoluble form in inclusion bodies. After lysis the washed inclusion bodies were solubilized and an l-arginine assisted protein refolding route was employed to obtain biologically active protein. rhBcl-2 was purified further by nickel chelate chromatography to give protein of >95% purity, with an overall yield of 5 mg per g of E. coli cell paste. Edman sequencing showed that approximately 90% of the rhBcl-2 retained the initiating methionine residue. Analytical size exclusion chromatography suggested that the refolded and purified rhBcl-2 was monomeric in nondenaturing solution. Purified protein had an affinity for a Bax BH3 domain peptide comparable to that for in vivo folded recombinant human Bcl-2 and suppressed caspase activation in a cell-free assay for apoptosis. 1H NMR spectroscopy of rhBcl-2, both free and complexed with the Bax BH3 domain peptide, provided further evidence for the structural and functional integrity of the refolded protein. These findings parallel and extend those of Muchmore et al., who found that a loop deletion mutant of human Bcl-XL retained anti-apoptotic function.     

Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.     

The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis

Ec DOS is a heme-based gas sensor enzyme that catalyzes conversion from cyclic-di-GMP to linear-di-GMP in response to gas molecules, such as oxygen, CO and NO. Ec DOS contains an N-terminal heme-binding PAS domain and C-terminal phosphodiesterase domain. Based on crystal structures of the isolated heme-binding domain, it is suggested that the FG loop is involved in intra-molecular signal transduction to the catalytic domain. We generated nine full-length proteins mutated at ionic and non-ionic polar residues between positions 83 and 96 corresponding to the F-helix and FG loop, and examined the heme binding properties, autoxidation rates, and catalytic activities of mutant proteins. N84A and R85A mutant proteins displayed lower heme binding affinities, consistent with the finding that Asn84 interacts with propionate of protoporphyrin IX, and Arg85 with Asp40 on the heme proximal side. Autoxidation rates (0.058-0.54 min⁻¹) of R91A, S96A and K89A/R91A/E93A mutant proteins were significantly higher than that (0.0053 min⁻¹) of wild-type protein, suggesting that these residues in the FG loop form heme distal architecture conferring stability to the Fe(II)-O₂ complex. Catalytic activities of N84A and R85A mutant proteins with low heme affinity were significantly higher than those of wild-type protein in the absence of gas molecules. Accordingly, we propose that loss of heme binding enhances basal catalysis without the gas molecule, consistent with previous reports on heme inhibition of Ec DOS catalysis.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Spectroscopic and functional characterization of nitrophorin 7 from the blood-feeding insect Rhodnius prolixus reveals an important role of its isoform-specific N-terminus for proper protein function

Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [KIII(eq)(NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10(9) M-1 --> 4.0 x 10(6) M-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1-4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [KIII(eq)(histamine)] of 10(5) M-1 compared to the histamine concentration of 1-10 x 10(-9) M in the blood. A major part of this study deals with the N-terminus, 1Leu-Pro-Gly-Glu-Cys5 of NP7, which is not found in NP1-4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SIGNALP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987-6994]. In marked contrast to wild-type NP7, NP7(Delta1-3) exhibits a very high NO affinity at pH 7.5 [KIII(eq)(NO) approximately 10(9) M-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the 1Leu-Pro-Gly3 peptide was removed. However, 1H NMR and EPR spectroscopy show that the electronic properties of the FeIII cofactor are similar in both wild-type NP7 and NP7(Delta1-3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Delta1-3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 A), suggests that interaction of the 1Leu-Pro-Gly3 peptide with the A-B and/or G-H loops is key for proper protein function.     

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.     

Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.     

Assignment of heme resonances and determination of the electronic structures of high- and low-spin nitrophorin 2 by 1H and 13C NMR spectroscopy: an explanation of the order of heme methyl resonances in high-spin ferriheme proteins

The (1)H NMR resonances of the heme substituents of the low-spin Fe(III) form of nitrophorin 2, as its complexes with N-methylimidazole (NP2-NMeIm) and imidazole (NP2-ImH), have been assigned by a combination of (1)H homonuclear two-dimensional NMR techniques and (1)H-(13)C HMQC. Complete assignment of the proton and partial assignment of the (13)C resonances of the heme of these complexes has been achieved. Due to favorable rates of ligand exchange, it was also possible to assign part of the (1)H resonances of the high-spin heme via saturation transfer between high- and low-spin protein forms in a partially liganded NP2-NMeIm sample; additional resonances (vinyl and propionate) were assigned by NOESY techniques. The order of heme methyl resonances in the high-spin form of the protein over the temperature range of 10-37 degrees C is 8 = 5 > 1 > 3; the NMeIm complex has 5 > 1 > 3 > 8 as the order of heme methyl resonances at <30 degrees C, while above that temperature, the order is 5 > 3 > 1 > 8, due to crossover of the closely spaced 3- and 1-methyl resonances of the low-spin complex at higher temperatures. This crossover defines the nodal plane of the heme orbital used for spin delocalization as being oriented 162 +/- 2 degrees clockwise from the heme N(II)-Fe-N(IV) axis for the heme in the B orientation. For the NP2-ImH complex, the order of heme methyl resonances is 3 > 5 > 1 > 8, which defines the orientation of the nodal plane of the heme orbital used for spin delocalization as being oriented approximately 150-155 degrees clockwise from the heme N(II)-Fe-N(IV) axis. In both low-spin complexes, the results are most consistent with the exogenous planar ligand controlling the orientation of the nodal plane of the heme orbital. In the high-spin form of NP2, the proximal histidine plane is shown to be oriented 135 degrees clockwise from the heme N(II)-Fe-N(IV) axis, again for the B heme orientation. A correlation between the order of heme methyl resonances in the high-spin form of NP2 and several other ferriheme proteins and an apparent 90 degrees shift in the nodal plane of the orbital involved in spin delocalization from that expected on the basis of the orientation of the axial histidine imidazole nodal plane have been explained in terms of bonding interactions between Fe(III), the axial histidine imidazole nitrogen, and the porphyrin pi orbitals of the high-spin protein.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.