Amyloid fibril formation by A beta 16-22, a seven-residue fragment of the Alzheimer's beta-amyloid peptide, and structural characterization by solid state NMR

The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH(2), called A beta(16-22) and representing residues 16-22 of the full-length beta-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in A beta(16-22) fibrils are investigated by solid state (13)C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a beta-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit (13)C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional (13)C-(13)C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain (13)C NMR signals and indicates that the beta-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. (13)C multiple-quantum (MQ) NMR and (13)C/(15)N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of beta-sheets in the A beta(16-22) fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of beta-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. A beta(16-22) is among the shortest fibril-forming fragments of full-length beta-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.     

The interaction of beta-amyloid protein fragment (12-28) with lipid environments.

The neurotoxicity of beta-amyloid protein (beta AP) fragments may be a result of their solution conformation, which is very sensitive to solution conditions. In this work we describe NMR and CD studies of the conformation of beta AP(12-28) in lipid (micelle) environments as a function of pH and lipid type. The interaction of beta AP(12-28) with zwitterionic dodecylphosphocholine (DPC) micelles is weak and alters the conformation when compared to water solution alone. By contrast, the interaction of the peptide with anionic sodium dodecylsulfate (SDS) micelles is strong: beta AP(12-28) is mostly bound, is alpha-helical from K16 to V24, and aggregates slowly. The pH-dependent conformation changes of beta AP(12-28) in solution occur in the pH range at which the side-chain groups of E22, D23, H13, and H14 are deprotonated (pKas ca. 4 and 6.5); the interaction of beta AP(12-28) with SDS micelles alters the pH-dependent conformational transitions of the peptide whereas the weak interaction with DPC micelles causes little change.     

Beta-amyloid peptide toxicity in organotypic hippocampal slice culture involves Akt/PKB, GSK-3beta, and PTEN

In the present study we investigated the toxicity induced by exposing organotypic slice culture to beta-amyloid peptide 25-35 (25microM) for 1, 3, 6, 12, 24 and 48h. To elucidate a mechanism involved in its toxicity, we studied the PI3-K cell signaling pathway, particularly Akt/PKB, GSK-3beta, and PTEN proteins. Cell death was quantified by propidium iodide uptake and proteins were analyzed by immunoblotting. Our results showed a significant cell death after 48h of beta-amyloid 25-35 peptide exposition. The exposition of cultures to beta-amyloid peptide resulted in an increase in the phosphorylation state of Akt and GSK-3beta proteins after 6h, followed by a decrease of the phosphorylation state of these proteins after 12h of exposition. However, after 24h of peptide treatment, the phosphorylation of GSK-3beta presented a new increase while the phosphorylation of Akt remained down. The immunocontent of the PTEN protein, an indirect Akt phosphatase, increased after 24 and 48h of beta-amyloid exposition. These results suggest an involvement of Akt dephosphorylation/inactivation in the toxicity induced by the beta-amyloid 25-35 peptide in organotypic slice hippocampal culture, probably induced by increasing PTEN immunocontent. Taken together, our results provide more information about the molecular mechanisms involved on beta-amyloid peptide toxicity.     

Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance

We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (A beta(1-40)) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between (13)C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional (13)C magic-angle spinning NMR spectra of the labeled A beta(1-40) samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 +/- 0.5 A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly beta-sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A beta(1-40), including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length beta-amyloid fibrils.     

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.     

Chronic nicotine treatment reduces beta-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)

Alzheimer's disease neuropathology is characterised by beta-amyloid plaques and neurofibrillary tangles. Inhibition of beta-amyloid accumulation may be essential for effective therapy in Alzheimer's disease. In this study we have treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein [Tg(Hu.APP695.K670N-M671L)2576], which develop brain beta-amyloid deposits, with nicotine in drinking fluid (200 microg/mL) from 9-14.5 months of age (5.5 months). A significant reduction in amyloid beta peptide 1-42 positive plaques by more than 80% (p < 0.03) was observed in the brains of nicotine treated compared to sucrose treated transgenic mice. In addition, there was a selective reduction in extractable amyloid beta peptides in nicotine treated mice; cortical insoluble 1-40 and 1-42 peptide levels were lower by 48 and 60%, respectively (p < 0.005), whilst there was no significant change in soluble 1-40 or 1-42 levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce amyloid beta peptide aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in Alzheimer's disease.     

The Alzheimer beta-peptide shows temperature-dependent transitions between left-handed 3-helix, beta-strand and random coil secondary structures

The temperature-induced structural transitions of the full length Alzheimer amyloid beta-peptide [A(beta)(1-40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 3(1) (polyproline II; PII)-helix at 0 degrees C to a random coil state at 60 degrees C, with an average DeltaH of 6.8 +/- 1.4 kJ.mol(-1) per residue, obtained by fitting a Zimm-Bragg model to the CD data. The transition is noncooperative for the shortest N-terminal fragment A(beta)(1-9) and weakly cooperative for A(beta)(1-40) and the longer fragments. By analysing the temperature-dependent 3J(HNH(alpha)) couplings and hydrodynamic radii obtained by NMR for A(beta)(1-9) and A(beta)(12-28), we found that the structure transition includes more than two states. The N-terminal hydrophilic A(beta)(1-9) populates PII-like conformations at 0 degrees C, then when the temperature increases, conformations with dihedral angles moving towards beta-strand at 20 degrees C, and approaches random coil at 60 degrees C. The residues in the central hydrophobic (18-28) segment show varying behaviour, but there is a significant contribution of beta-strand-like conformations at all temperatures below 20 degrees C. The C-terminal (29-40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of A(beta) may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.     

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.     

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.     

Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.

Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Conformational studies of the C-terminal 16-amino-acid-residue fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

The structure and stability of the 16-amino-acid-residue fragment [IG(46-61)] corresponding to the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus was investigated by means of CD and NMR spectroscopy and by differential scanning calorimetry. The CD and 2D NMR experiments were carried out (i) in water at different temperatures and (ii) at one temperature (305 K), with only CD, at different TFE concentrations. Our results show that the IG(46-61) peptide possesses organized three-dimensional structure at all investigated temperatures. The three-dimensional structure of the IG(46-61) peptide resembles the general shape of a beta-hairpin that is also observed for this peptide in the experimental structure of the B3 domain in the whole G protein; the structure is stabilized by hydrophobic interactions between nonpolar side chains. Our study shows that the melting temperature of the IG(46-61) peptide is about 320 K which supports the hypothesis that the investigated peptide can serve as a folding initiation site of the B3 domain of the immunoglobulin binding protein G.     

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

Affinity-based inhibition of beta-amyloid toxicity

Strategies for interfering with protein aggregation are important for elucidating and controlling the pathologies of amyloid diseases. We have previously identified compounds that block the cellular toxicity of the beta-amyloid peptide, but the relationship between their ability to inhibit toxicity and their affinity for A beta is unknown. To elucidate this relationship, we have developed an assay capable of measuring the affinities of small molecules for beta-amyloid peptide. Our approach employs immobilized beta-amyloid peptide at low density to minimize the problems that arise from variability in the beta-amyloid aggregation state. We found that low-molecular weight (MW of 700-1700) ligands for beta-amyloid can be identified readily by using surface plasmon resonance. The best of these bound effectively (K(d) approximately 40 microM) to beta-amyloid. The affinities measured for peptides in the SPR assay correspond to results from A beta cell toxicity assays. The most potent ligands for immobilized beta-amyloid are the most potent inhibitors of the neuronal cell toxicity of beta-amyloid. Compounds with dissociation constants above approximately 100 microM did not show significant activity in the cell toxicity assays. Our data support the hypothesis that ligands exhibiting greater affinity for the beta-amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity.     

Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR

Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Kinetics of aggregation of synthetic beta-amyloid peptide.

beta-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimer's disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-beta conformation. beta-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of beta-amyloid peptide, beta(1-40), was synthesized and characterized. beta(1-40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that beta(1-40) contained approximately two-thirds beta-structure, but no alpha-helical character. Quasi-elastic and classical light scattering measurements showed that beta(1-40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 x 10(6) after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Epitope mapping of the phosphorylation motif of the HIV-1 protein Vpu bound to the selective monoclonal antibody using TRNOESY and STD NMR spectroscopy

The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.     

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.