Christopher J. Falzone

The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state.

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.

Characterization of taxol in methylene chloride by nmr spectroscopy

Abstract The 1 H and 13 C NMR spectra of taxol in methylene chloride were analyzed using two-dimensional methods. 13 C chemical shift assignments were made based on inverse-detected two-dimensional NMR experiments and are reported. The NOESY data collected in this solvent suggest that the structure in solution is similar to the X-ray structure of the taxol analogue taxotere.

Functional and structural characterization of the 2/2 hemoglobin from Synechococcus sp. PCC 7002.

Cyanobacterium Synechococcus sp. PCC 7002 contains a single gene (glbN) coding for GlbN, a protein of the 2/2 hemoglobin lineage. The precise function of GlbN is not known, but comparison to similar 2/2 hemoglobins suggests that reversible dioxygen binding is not its main activity. In this report, the results of in vitro and in vivo experiments probing the role of GlbN are presented. Transcription profiling indicated that glbN is not strongly regulated under any of a large number of growth conditions and that the gene is probably constitutively expressed. High levels of nitrate, used as the sole source of nitrogen, and exposure to nitric oxide were tolerated better by the wild-type strain than a glbN null mutant, whereas overproduction of GlbN in the null mutant background restored the wild-type growth. The cellular contents of reactive oxygen/nitrogen species were elevated in the null mutant under all conditions and were highest under NO challenge or in the presence of high nitrate concentrations. GlbN overproduction attenuated these contents significantly under the latter conditions. The analysis of cell extracts revealed that the heme of GlbN was covalently bound to overproduced GlbN apoprotein in cells grown under microoxic conditions. A peroxidase assay showed that purified GlbN does not possess significant hydrogen peroxidase activity. It was concluded that GlbN protects cells from reactive nitrogen species that could be encountered naturally during growth on nitrate or under denitrifying conditions. The solution structure of covalently modified GlbN was determined and used to rationalize some of its chemical properties.

Characterization of the heme-histidine cross-link in cyanobacterial hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the 1H, 15N, and 13C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N2-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal pKa whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.

Partial NMR assignments and secondary structure mapping of the isolated α subunit of Escherichia coli tryptophan synthase, a 29-kD TIM barrel protein

The α subunit of tryptophan synthase (αTS) from S. typhimurium belongs to the triosephosphate isomerase (TIM) or the (β/α)8 barrel fold, one of the most common structures in biology. To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29‐kD αTS by using standard heteronuclear multidimensional NMR methods on uniformly 15N‐ and 15N/13C‐labeled protein and on protein selectively 15N‐labeled at key hydrophobic residues. The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen‐deuterium (H‐D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E. coli αTS. Because most of the amide protons that are slow to exchange with solvent correspond to the β‐sheet residues, the β‐barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E. coli αTS. A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

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.

NMR assignments for the aldopentoses

Abstract The 1 H and 13 C NMR assignments for the simple aldopentopyranoses in 2 H 2 O were made using high field 2D NMR spectroscopy. The furanose forms of ribose were also tentatively assigned. The 2D techniques used to assign the resonances include the HMQC, HMBC, 2QF-COSY, TOCSY, 2Q, and INADEQUATE experiments. Selective 1D TOCSY experiments were also necessary to assign the crowded proton spectrum of ribose.

Where U and I meet.

A detailed NMR description of the compact unfolded state of an unstable protein reveals the existence of native and non-native structure under non-denaturing conditions. The structural characterization of this and similar species may provide clues about protein folding mechanisms.

Chemical Rescue of Site-Modified Ligands to the Iron-Sulfur Clusters of Psac In Photosystem I

The PsaC subunit of Photosystem I (PS I) is an 8.6 kDa protein which contains binding sites for two [4Fe-4S] clusters. To investigate structure-function relationships in PsaC we changed cysteine 14, which ligates FB, to glycine [1]. After cluster reconstitution unbound PsaC showed an EPR spectrum consistent with the presence of two [4Fe-4S] clusters, one S = 1/2 and the other S = 3/2. We proposed that 2-mercaptoethanol, a reagent used in the reconstitution protocol, serves as an external rescue ligand in the absence of a biological ligand to the fourth iron [1]. Instead of the expected formation of a [3Fe-4S] cluster in the glycine mutants, chemical rescue allows for the formation of a [4Fe-4S] cluster. The present work is aimed at obtaining direct evidence to support the chemical rescue hypothesis. We reconstituted the C14G/C34S mutant of PsaC with 4-fluorothiophenol or 2,2,2-tlifluoroethanethiol as the external thiolate ligand. Successful insertion of [4Fe-4S] clusters was confirmed by electron paramagnetic resonance (EPR) spectroscopy. Flourine-19 nuclear magnetic resonance (19F NMR) spectroscopy showed paramagnetically shifted resonances that were attributed to cluster-bound ligand.