We describe here details of the hydrogen-deuterium (H/D) exchange behavior of the Alzheimer's peptide Abeta(1)(-)(40), while it is a resident in the amyloid fibril, as determined by high-resolution solution NMR. Kinetics of H/D exchange in Abeta(1)(-)(40) fibrils show that about half the backbone amide protons exchange during the first 25 h, while the other half remain unexchanged because of solvent inaccessibility and/or hydrogen-bonded structure. After such a treatment for 25 h with D(2)O, fibrils of (15)N-enriched Abeta were dissolved in a mixture of 95% dimethyl sulfoxide (DMSO) and 5% dichloroacetic acid (DCA) and successive heteronuclear (1)H-(15)N HSQC spectra were collected to identify the backbone amides that did not exchange in the fibril. These studies showed that the N and C termini of the peptide are accessible to the solvent in the fibril state and the backbone amides of these residues are readily exchanged with bulk deuterium. In contrast, the residues in the middle of the peptide (residues 16-36) are mostly protected, suggesting that that many of the residues in this segment of the peptide are involved in a beta structure in the fibril. Two residues, G25 and S26, exhibit readily exchangeable backbone amide protons and therefore may be located on a turn or a flexible part of the peptide. Overall, the data substantially supports current models for how the Abeta peptide folds when it engages in the amyloid fibril structure, while also addressing some discrepancies between models.

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The three-dimensional structure of an N-terminal fragment comprising the first 51 amino acids from human plasma vitronectin, the somatomedin B (SMB) domain, has been determined by two-dimensional NMR approaches. An average structure was calculated, representing the overall fold from a set of 20 minimized structures. The core residues (18 -41) overlay with a root mean square deviation of 2.29 ؎ 0.62 Å. The N-and Cterminal segments exhibit higher root mean square deviations, reflecting more flexibility in solution and/or fewer long-range NOEs for these regions. Residues 26 -30 form a unique single-turn ␣-helix, the locus where plasminogen activator inhibitor type-1 (PAI-1) is bound. This structure of this helix is highly homologous with that of a recombinant SMB domain solved in a co-crystal with PAI-1 (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544), although the remainder of the structure differs. Significantly, the pattern of disulfide cross-links observed in this material isolated from human plasma is altogether different from the disulfides proposed for recombinant forms. The NMR structure reveals the relative orientation of binding sites for cell surface receptors, including an integrin-binding site at residues 45-47, which was disordered and did not diffract in the co-crystal, and a site for the urokinase receptor, which overlaps with the PAI-1-binding site.Human vitronectin is a glycoprotein found in the circulation, where it contributes to hemostasis by regulating blood coagulation and fibrinolysis (1, 2). Vitronectin is also found in the ECM, 1 where it plays important roles in cell adhesion, pericellular proteolysis, tissue invasion, angiogenesis, and metastasis (3-7). The variety of functions of vitronectin in the two microenvironments is a manifestation of its ability to interact with numerous humoral and cellular proteins. An important binding partner for vitronectin is the serine protease inhibitor PAI-1, which also is found both in circulation and the ECM. Furthermore, it has different activities depending upon this localization; the anti-protease activity of PAI-1 that regulates thrombolysis in the circulation is targeted instead toward pericellular proteolysis when localized to the ECM or cell/matrix boundary. Vitronectin binds to PAI-1 with high affinity and stabilizes the inhibitor in its active conformation (8, 9). Vitronectin can also associate with PAI-1 and assemble to form higher order complexes (10) that exhibit altered adhesive functions (11). Key to the adhesive functions of vitronectin are its interactions with cell-surface receptors including integrins and uPAR.A widely accepted model suggests that vitronectin is organized into functional domains that provide the broad repertoire necessary for binding to target ligands (12)(13)(14). We recently used computational methods to predict the structure of the three domains comprising vitronectin (15). A threading algorithm gave high confidence predictions for the central domai...

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Vitronectin is a large glycoprotein with wide ranging distribution and function. The hallmark feature of the vitronectin structure is a series of distinct functional domains that allow it to interact both with itself and with a number of other ligands in a variety of environments including the circulation, extracellular matrix, and platelets (1-4). Of particular interest is the N-terminal somatomedin B (SMB) 1 domain of vitronectin. This domain contains the high affinity binding site for the serpin PAI-1. The interaction between PAI-1 and vitronectin is important to the function of both proteins in thrombolysis, cell adhesion, and pericellular proteolysis (1, 5-9). 2 Equally important for the adhesive properties of vitronectin are binding sites for cell-surface receptors, including integrins and uPAR, that are housed within this small N-terminal domain (2, 10 -15).Because it is known that the SMB domain provides a high affinity binding site for PAI-1 and that this interaction stabilizes PAI-1 in its physiologically active form, the structure of the SMB domain of vitronectin has been hotly pursued. Computational predictions for the structure of this domain using a threading algorithm were challenging compared with other domains from vitronectin (16), as there were no reported structures at the time for homologues of this small domain containing four disulfides. Nevertheless, there are over 100 homologues in the sequence data base, suggesting that this folding motif has been conserved in evolution. Only recently have three-dimensional structures describing the SMB domain become available from two different approaches. First, an x-ray structure has been reported on a co-crystal of PAI-1 and a recombinant form of the SMB domain expressed in Escherichia coli (17). Subsequently, we completed the determination of a solution structure for the SMB domain purified from circulating vitronectin that was isolated from human plasma (18). Although the two structures differ in overall fold, they share a common feature, a single ␣-helix that contains key amino acids for PAI-1 binding. Not surprisingly, a recent report using NMR on a recombinant SMB arrived at a similar structure to that observed in the co-crystal with PAI-1 (19).Key to understanding the structure of the SMB domain is defining the correct dis...

Gold electrodes modified with a DNA 20‐mer via a thiol linkage were shown to efficiently capture the complementary 20‐mer that was tagged with anthraquinone (AQ) reporter groups. Treatment of the electrodes with 2‐mercaptoethanol was required for both efficient surface hybridization and for reversible electron transfer between the gold electrode and the tethered anthraquinone tag. In addition, slow thermal annealing through the melting temperature of the duplex increased the efficiency of the process. Electrodes that had been modified with a ds‐DNA bearing AQ tags gave slightly greater voltammetric signals, but after dissociation of the duplex were not as effective as the ss‐DNA in capturing the complementary 20‐mer from the buffer solutions.