Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody "imprint" of the Cyt b surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 phox subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phagedisplay analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29 TAGRF 33 and 183 PQVNPI 188 from discontinuous regions of p22 phox . Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.Human phagocyte flavocytochrome b (Cyt b) 1 is an electron transferase that directs metabolic electrons across the plasma membrane to reduce molecular oxygen and form superoxide (O 2 . ). The production of superoxide by phagocytes, which involves modulation of Cyt b structure, is essential for antimicrobial host defense (1), plays a central role in inflammatory tissue injury (2), and may play a more general role in cellular regulation (3). Individuals with a defective Cyt b have insufficient superoxide production, suffer from chronic granulomatous disease (4), and sustain repeated life-threatening infections (5). Despite its cardinal role in many inflammatory processes and possibly in growth regulation, few experimental methods have been able to provide information on the structure of Cyt b. Therefore, we have developed an alternative method to describe topological features of the Cyt b surface. Because antibodies and their cognate antigens form complementary surfaces of interaction (6), we have sought structural information about Cyt b from an antibody binding site, specific for the Cyt b surface. We recently reported mapping of monoclonal antibody epitopes and functional sites of Cyt b, using random sequence phage-display peptide libraries (7;8). mAb 44.1 recognizes Cyt b in situ in permeabilized human neutrophils and thus contains information about the three-dimensional structure of the protein sur...

Six independent lines of evidence point to the existence of heme-containing compounds and͞or hemoglobin breakdown products in extracts of trabecular tissues of the large theropod dinosaur Tyrannosaurus rex. These include signatures from nuclear magnetic resonance and electron spin resonance that indicate the presence of a paramagnetic compound consistent with heme. In addition, UV͞visible spectroscopy and high performance liquid chromatography data are consistent with the Soret absorbance characteristic of this molecule. Resonance Raman profiles are also consistent with a modified heme structure. Finally, when dinosaurian tissues were extracted for protein fragments and were used to immunize rats, the resulting antisera reacted positively with purified avian and mammalian hemoglobins. The most parsimonious explanation of this evidence is the presence of blood-derived hemoglobin compounds preserved in the dinosaurian tissues.