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The cardiac hormone B-type natriuretic peptide (BNP) is synthesized as a prepro 134 residue molecule which is further proteolytically processed into a 76 residue fragment termed N-terminal proBNP (NT-proBNP) and the active portion of this hormone, a 32-residue disulfide-linked peptide (BNP-32). The active hormone regulates cardiac hemodynamic output while as yet no biological function has been attributed to NT-proBNP. Some solution properties of synthetically generated NT-proBNP in benign media are known. The protein is monomeric, elutes aberrantly on size-exclusion chromatography as an apparent larger molecular species, and possesses little global secondary structure as assessed by circular dichroism. To explore the solution structure of NT-proBNP in greater detail, we use 2D-NOESY and 2D-TOCSY NMR on recombinant NT-proBNP to obtain a high resolution solution conformation at the alpha-carbon level. Importantly, NH(i)-NH(i+1) coupling is virtually absent at room temperature implying that large stretches of primary sequence are unordered. Together, the results of these physicochemical measurements classify NT-proBNP as a naturally unfolded protein referred to as an Intrinsically Unstructured Protein (IUP). The calculations of FoldIndex, a computer program which predicts disorder, were compared to the experimental results described here for NT-proBNP in addition to proBNP. NT-proBNP thus appears to be an ideal candidate for the study of native, unfolded proteins.

Matrix metalloproteinases (MMPs) function in homeostatic and repair processes, but unregulated catalysis by these extracellular proteinases leads to the pathological destruction of tissue proteins. An important mechanism for controlling enzyme activity might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase system of phagocytes. We have shown that inactivation of MMP-7 (matrilysin) by HOCl coincides with the formation of a novel oxidation product, WG-4, through modification of adjacent tryptophan and glycine residues and loss of 4 atomic mass units. Here, we use mass spectrometry, UV/visible spectroscopy, hydrogen-deuterium exchange, and NMR spectroscopy to investigate the formation and structure of WG-4. For the initial step, HOCl chlorinates the indole ring of tryptophan. The resulting 3-chloroindolenine generates a previously unknown cyclic indole-amide species, in which tryptophan cross-links to the main chain nitrogen of the adjacent glycine residue to form an aromatic six-membered ring. WG-4 kinks and stiffens the peptide backbone, which may hinder the interaction of substrate with the catalytic pocket of MMP-7. Our observations indicate that specific structural motifs are important for controlling protein modification by oxidants and suggest that pericellular oxidant production by phagocytes might limit MMP activity during inflammation.

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(±0.09) A. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the β-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the 15 N T 1 , T 2 and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T 2 derived apparent backbone exchange rate and amide 1 H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.

2,6-Dimethoxyphenylphosphirane is readily converted to its oxide and sulfide whose pyrolysis and (perhaps) photolysis lead to the generation of phosphinidene chalcogenides Ar–P Z (Z=O,S). Trapping experiments were carried out under conditions similar to the kinetic studies of the phosphirane chalcogenide pyrolyses that confirmed the formation of free Ar–P Z. The trapping experiments were in accord with carbene-like character for Ar–P Z, and the activation parameters suggest a non-least motion pathway for the addition of Ar–P Z to olefins. This represents quantitative evidence for the validity of the predictions of frontier-orbital theory for species that undergo reactions with small (or no) enthalpic barriers.

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13 C, 15 N-apo-CRBP II and 15 N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/ residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A ˚ . Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the bC-bD turn and the bE-bF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the bC-bD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligandbinding pocket. Residues 28-35, which form the second helix of the helixturn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the bE-bF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1 H exchange rates and 15 N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the ms to ms timescale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15 N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the ms to ms time-scale which result in increased access to the binding cavity. # 1999 Academic Press

The solution structures of a duplex DNA dodecamer containing a cis-syn cyclobutane thymine dimer d(GCACGAAT[ cs ]TAAG)·d(CTTAATTCG TGC) and its native parent sequence were determined using NMR data collected at 750 MHz. The dodecamer sequence corresponds to the section of a site-specific cis-syn dimer containing 49-mer that was found to be the binding site for the dimer-specific T4 denV endonuclease V repair enzyme by chemical and enzymatic footprinting experiments. Structures of both sequences were derived from NOE restrained molecular dynamics/simulated annealing calculations using a fixed outer layer of water and an inner dynamic layer of water with sodium counterions. The resulting structures reveal a subtle distortion to the phosphodiester backbone in the dimer-containing sequence which includes a B II phosphate at the T9pA10 junction immediately 3′ to the dimer. The B II phosphate, established experimentally by analysis of the 31 P chemical shifts and interpretation of the 3 J P-H3′ values using an optimized Karplus relationship, enables the DNA helix to accommodate the dimer by destacking the base 3′ to the dimer. Furthermore, the structures provide explanations for the unusually shifted T8-N3H imino, A16-H2 and T8-Me proton resonances and T9pA10 31 P NMR resonance and are consistent with bending, unwinding, and thermodynamic data. The implications of the structural data for the mechanism by which cis-syn dimers are recognized by repair enzymes and bypassed by DNA polymerases are also discussed.