Polymerization of the amyloid beta (A␤) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected A␤-fragments containing the A␤ 16 -20 sequence, previously shown essential for A␤-A␤ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was A␤ 14 -23 . Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from A␤ 1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of A␤ 14 -23 oligomers in an antiparallel ␤-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of A␤-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

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We have previously shown that short peptides incorporating the sequence KLVFF can bind to the ϳ40-amino acid residue Alzheimer amyloid ␤-peptide (

The protein mediator MIF has been identified as being released from immune cells by glucocorticoid stimulation and to counter-regulate glucocorticoid action. MIF also has been described recently to exhibit dopachrome tautomerase activity and to be structurally homologous to the bacterial enzymes 4-oxalocrotonate tautomerase (4-OT) and 5-carboxymethyl-2-hydroxymuconate isomerase (CHMI). We performed site-directed mutagenesis and biochemical analyses of mouse MIF in order to identify amino acid residues and protein domains that are essential for enzymatic reactivity. Mutant proteins which lacked a free N-terminal proline residue were enzymatically inactive, as was a preparation of native MIF modified covalently at its N terminus by 3-bromopyruvate, suggesting that this proline has a catalytic function. Substitutions of the internal histidine residues 42 and 63 did not affect enzymatic activity, indicating that these basic residues are not involved in dopachrome tautomerization. Carboxy-truncated forms of MIF (residues 1-110 and 1-104) also were inactive, affirming the role of the carboxy terminus in stable trimer formation and the importance of the trimer for enzymatic activity. Additional evidence for the homotrimeric structure of MIF under native solution conditions was obtained by SDS-PAGE analysis of MIF after chemical cross-linking at low protein concentrations. The enzymatic activity of MIF was found to be reversibly inhibited by micromolar concentrations of fatty acids with chain lengths of at least 16 carbon atoms. Of note, molecular modeling of the substrate L-dopachrome methyl ester into the active site of MIF suggests an acid-catalyzed enzymatic mechanism that is different from that deduced from studies of the enzymes 4-OT and CHMI. Finally, in vitro analysis of an enzymatically inactive MIF species (P2 --> S) indicates that the glucocorticoid counter-regulatory activity of MIF can be functionally dissociated from its tautomerization activity.

Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 Å to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques.normal mode analysis ͉ statistical mechanics ͉ protein dynamics ͉ quasielastic neutron scattering P rotein domain motions are critical for proteins to coordinate precise biological functions. For example, coupled domain motions occur in genome regulatory proteins, motor proteins, signaling proteins, and structural proteins (1-6). Structural studies have documented the conformational flexibility in proteins accompanying their activities (7). Results from macroscopic studies, such as biochemical kinetics and single molecule detection studies, have also shown the importance of conformational dynamics and Brownian thermal fluctuations within proteins (5,(8)(9)(10). However, the time-dependent, dynamic processes that facilitate protein domain rearrangements remain poorly understood.The function of DNA polymerase I from Thermus aquaticus (Taq polymerase) (see Fig. 1) requires coordinated domain and subdomain motions within this protein to generate a precise ligatable nick on a DNA duplex (11-13). Taq polymerase performs nucleotide replacement reactions in DNA repair and RNA primer removal in DNA replication (14). During such processes, Taq polymerase utilizes a DNA polymerase domain to catalyze the addition of dNTP to the 3Ј hydroxyl terminus of an RNA primer and a 5Ј nuclease domain to cleave the downstream, single-stranded 5Ј nucleotide displaced by the growing upstream strand (11). Because the structure of Taq polymerase possesses an extended conformation with the polymerase and the 5Ј nuclease active sites separated by Ϸ70 Å (15-17), the DNA needs to be shuttled between these two distant catalytic sites when switching from the DNA synthesis mode to the nucleotide cleavage mode. This scenario is similar to that which occurs when the DNA needs to be shifted from the polymerase active site to the 3Ј-5Ј exonucleas...