Recent evidence suggests that amyloidogenic oligomers may be the toxic species in cell cultures. Thus, it is crucial to understand their structure and oligomerization mechanism in atomistic detail. By employing tens of fast central processing units and an advanced phase-space sampling algorithm, parallel-tempering molecular dynamics, we have explored the energy landscape of amyloidogenic peptide oligomerization in explicit water. A pentapeptide, DFNKF, derived from human calcitonin and its mutant, DFAKF, was simulated with a total simulation time of Ϸ500 ns. The detailed oligomerization process of a DFNKF parallel ␤-sheet formation at 300 K has been characterized. The assembly of a parallel ␤-sheet from the amorphous state mainly occurs via a ''bottleneck'' channel where the interstrand Asn-Asn stacking is the major interaction. The interactions of Asn-Asn stacking include both backbone and side-chain hydrogen bonds. The Asn-Asn interactions work like ''glue'' by sticking the DFNKF strands together and assist the ''on-pathway'' oligomerization. The Asn-Asn stacking observed here is similar to the Asn ladder commonly found in globular ␤-helix proteins. A control run shows that when Asn is mutated to Ala, the stability and population of the DFAKF parallel ␤-sheet is decreased. Furthermore, our in vitro mutagenesis experiments show that the ability of DFAKF peptides to form amyloid fibrils is significantly reduced, in agreement with the simulations. Knowledge of the energy landscape of oligomerization may provide hints for rational drug design, preventing amyloid-associated diseases.amyloid oligomerization ͉ bottleneck channel ͉ ␤-helix ͉ human calcitonin ͉ mutagenesis P roteins fold in vivo into their unique 3D structures to perform their biological functions. Yet, under certain conditions, proteins can misfold and aggregate, leading to the malfunctioning of the organism (1). Amyloid fibrosis is one such example, involving severe diseases such as Alzheimer's disease, Prion disorder, and Parkinson's disease (1). Although amyloidogenic diseases are characterized by the deposition of insoluble amyloid fibrils, recent evidence suggests that amyloid oligomers and protofibrils may actually be the toxic agents (2, 3). The increasing appreciation of the importance of the toxic oligomers in the pathological process has attracted considerable attention, focusing not only on their structures but also on the details of their oligomerization mechanism (4-6).X-ray diffraction data indicate that amyloid fibrils from peptides of unrelated origins have a similar cross-␤-fibril organization (7). Also, amyloidogenic proteins do not share sequence or structural homology. The noncrystallinity and insolubility of amyloid fibrils complicate the determination of their structures in atomic detail by conventional methods, such as x-ray crystallography and solution NMR. Nevertheless, the mechanisms of formation and the structure of amyloid fibrils are starting to be revealed by a number of experimental (8-10) and molecularmodeling methods (...

In this study, we used TD-PBE0 calculations to investigate the first singlet excited state (S(1)) behavior of 2-(2'-hydroxyphenyl)benzimidazole (HBI) and its amino derivatives. We employed the potential energy surfaces (PESs) at the S(1) state covering the normal syn, tautomeric (S(1)-T(syn)), and intramolecular charge-transfer (S(1)-T(ICT)) states in ethanol and cyclohexane to investigate the reaction mechanisms, including excited-state intramolecular proton transfer (ESIPT) and intramolecular charge-transfer (ICT) processes. Two new S(1)-T(ICT) states, stable in ethanol and cyclohexane, were found for HBI and its amino derivatives; they are twisted and pyramidalized. The flat PES of the ICT process makes the S(1)-T(ICT) states accessible. The S(1)-T(ICT) state is effective for radiationless relaxation, which is responsible for quenching the fluorescence of the S(1)-T(syn) state. In contrast to the situation encountered conventionally, the S(1)-T(ICT) state does not possess a critically larger dipole moment than its precursor, S(1)-T(syn) state; hence, it is not particularly stable in polar solvents. On the basis of the detailed PESs, we rationalize various experimental observations complementing previous studies and provide insight to understand the excited-state reaction mechanisms of HBI and its amino derivatives.

Utilizing concepts of protein building blocks, we propose a de novo computational algorithm that is similar to combinatorial shuffling experiments. Our goal is to engineer new naturally occurring folds with low homology to existing proteins. A selected protein is first partitioned into its building blocks based on their compactness, degree of isolation from the rest of the structure, and hydrophobicity. Next, the protein building blocks are substituted by fragments taken from other proteins with overall low sequence identity, but with a similar hydrophobic/hydrophilic pattern and a high structural similarity. These criteria ensure that the designed protein has a similar fold, low sequence identity, and a good hydrophobic core compared with its native counterpart. Here, we have selected two proteins for engineering, protein G B1 domain and ubiquitin. The two engineered proteins share ∼20% and ∼25% amino acid sequence identities with their native counterparts, respectively. The stabilities of the engineered proteins are tested by explicit water molecular dynamics simulations. The algorithm implements a strategy of designing a protein using relatively stable fragments, with a high population time. Here, we have selected the fragments by searching for local minima along the polypeptide chain using the protein building block model. Such an approach provides a new method for engineering new proteins with similar folds and low homology.

Increasing evidence suggests that amyloids and parallel beta helices may share similar motifs. A systemic analysis of beta helices is performed to examine their sequence and structural characteristics. Ile prefers to occur in beta strands. In contrast, Pro is disfavored, compatible with the underlying assumption in Pro-scanning mutagenesis. Cys, Asn, and Phe form significant homostacking (identical amino acid interactions). Asn is highly conserved in the high-energy, left-handed alpha-helical conformation, where it frequently forms amide stacking. Based on the observed prominent stacking of chemically similar residues in parallel beta helices, we propose that within the "cross-beta" framework, amyloids with longer peptide chains may have common structural features of in-register, parallel alignment, with the side chains forming identical amino acid ladders. The requirement of ladder formation limits the combinations of side chain interactions. Such a limit combined with environmental conditions (e.g., pH, concentration) could be a major reason for the ability of most polypeptides to form amyloids.