Andrij Baumketner

Amyloid β-protein monomer structure: A computational and experimental study

The structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimers disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Aβ42 peptide conformations are dominated by loops and turns but show some helical structure in the C‐terminal hydrophobic tail. A second calculation on Aβ42 in a solvent‐free environment yields compact structures turned “inside out” from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Aβ42 −3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross‐sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent‐free family of structures. Implications of the calculations on the early stages of aggregation of Aβ42 are discussed.

Structure of the 21-30 fragment of amyloid β-protein

Folding and self‐assembly of the 42‐residue amyloid β‐protein (Aβ) are linked to Alzheimers disease (AD). The 21–30 region of Aβ, Aβ(21–30), is resistant to proteolysis and is believed to nucleate the folding of full‐length Aβ. The conformational space accessible to the Aβ(21–30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the “native” state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24–K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non‐native states of this peptide are compact and retain a native‐like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape.

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T<T(f)). For confinement to positively impact folding rates under physiological conditions, it is hence necessary for the protein to exhibit a folding transition above the temperature at which it exhibits its fastest folding rate (T(m)<T(f)). We designed a protein with this property by reducing the energetic frustration in the original alpha/beta sandwich substrate protein. The modified protein exhibited a twofold acceleration in folding rates upon encapsulation. This rate enhancement is due to a mechanistic change in folding involving the elimination, upon encapsulation, of accessible local energy minima corresponding to structures with large radii of gyration. For this protein, confinement hence plays more than the role of a passive cage, but rather adopts an active role, accelerating folding rates by decreasing the roughness of the energy landscape of the protein.

Effects of Familial Alzheimer's Disease Mutations on the Folding Nucleation of the Amyloid β-Protein

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimers disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid beta-protein (Abeta) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Abeta. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Abeta. Enhanced aggregation in Abeta with familial Alzheimers disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Abeta. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Abeta folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Abeta are fundamentally different.

Role of the familial Dutch mutation E22Q in the folding and aggregation of the 15–28 fragment of the Alzheimer amyloid-β protein

Amyloid fibrils, large ordered aggregates of amyloid β proteins (Aβ), are clinical hallmarks of Alzheimers disease (AD). The aggregation properties of amyloid β proteins can be strongly affected by single-point mutations at positions 22 and 23. The Dutch mutation involves a substitution at position 22 (E22Q) and leads to increased deposition rates of the AβE22Q peptide onto preseeded fibrils. We investigate the effect of the E22Q mutation on two key regions involved in the folding and aggregation of the Aβ peptide through replica exchange molecular dynamics simulations of the 15–28 fragment of the Aβ peptide. The Aβ15–28 peptide encompasses the 22–28 region that constitutes the most structured part of the Aβ peptide (the E22–K28 bend), as well as the central hydrophobic cluster (CHC) (segment 17–21), the primary docking site for Aβ monomers depositing onto fibrils. Our simulations show that the 22–28 bend is preserved in the Aβ(15–28) peptide and that the CHC, which is mostly unstructured, interacts with this bend region. The E22Q mutation does not affect the structure of the bend but weakens the interactions between the CHC and the bend. This leads to an increased population of β-structure in the CHC. Our analysis of the fibril elongation reaction reveals that the CHC adopts a β-strand conformation in the transition state ensemble, and that the E22Q mutation increases aggregation rates by lowering the barrier for Aβ monomer deposition onto a fibril. Thermodynamic signatures of this enhanced fibrillization process from our simulations are in good agreement with experimental observations.

Amyloid β-protein: Experiment and theory on the 21-30 fragment

The structure of the 21-30 fragment of the amyloid beta-protein (Abeta) was investigated by ion mobility mass spectrometry and replica exchange dynamics simulations. Mutations associated with familial Alzheimers disease (E22G, E22Q, E22K, and D23N) of Abeta(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the WT peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding. Results for the mutants show that substitutions at E22 do little to alter the overall structure of the fragment. A substitution at D23 resulted in a change of structure for Abeta(21-30). A comparison of these gas-phase studies to previous solution-phase studies reveals that the peptide can fold in the absence of solvent to a structure also seen in solution, highlighting the important role of the D23 hydrogen bonding network in stabilizing the fragments folded structure.

Stability of a protein tethered to a surface

Surface-tethered proteins are increasingly being used in a variety of experimental situations, and they are the basis for many new technologies. Nevertheless, a thorough understanding of how a surface can impact the native state stability of an attached protein is lacking. In this work, the authors use molecular dynamics simulations of a model beta-barrel protein to investigate how surface tethering influences native state stability. They find that stability, as measured by the folding temperature Tf, can be either increased, decreased, or remain unchanged as a result of tethering. Observed shifts are highly dependent on the location of residue used as the tether point, and stability is influenced by a number of factors, both energetic and entropic. These factors include native state vibrations, loss of bulk unfolded conformations, changes to the unfolded state ensemble, and the emergence of an entropic term not present for the bulk protein. They discuss each of these contributions in detail and comment on their relative importance and connection to experiment.

An image-based reaction field method for electrostatic interactions in molecular dynamics simulations of aqueous solutions.

In this paper, a new solvation model is proposed for simulations of biomolecules in aqueous solutions that combines the strengths of explicit and implicit solvent representations. Solute molecules are placed in a spherical cavity filled with explicit water, thus providing microscopic detail where it is most needed. Solvent outside of the cavity is modeled as a dielectric continuum whose effect on the solute is treated through the reaction field corrections. With this explicit/implicit model, the electrostatic potential represents a solute molecule in an infinite bath of solvent, thus avoiding unphysical interactions between periodic images of the solute commonly used in the lattice-sum explicit solvent simulations. For improved computational efficiency, our model employs an accurate and efficient multiple-image charge method to compute reaction fields together with the fast multipole method for the direct Coulomb interactions. To minimize the surface effects, periodic boundary conditions are employed for nonelectrostatic interactions. The proposed model is applied to study liquid water. The effect of model parameters, which include the size of the cavity, the number of image charges used to compute reaction field, and the thickness of the buffer layer, is investigated in comparison with the particle-mesh Ewald simulations as a reference. An optimal set of parameters is obtained that allows for a faithful representation of many structural, dielectric, and dynamic properties of the simulated water, while maintaining manageable computational cost. With controlled and adjustable accuracy of the multiple-image charge representation of the reaction field, it is concluded that the employed model achieves convergence with only one image charge in the case of pure water. Future applications to pKa calculations, conformational sampling of solvated biomolecules and electrolyte solutions are briefly discussed.

Aggregation of polyalanine in a hydrophobic environment

The dimerization of polyalanine peptides in a hydrophobic environment was explored using replica exchange molecular dynamics simulations. A nonpolar solvent (cyclohexane) was used to mimic, among other hydrophobic environments, the hydrophobic interior of a membrane in which the peptides are fully embedded. Our simulations reveal that while the polyalanine monomer preferentially adopts a beta-hairpin conformation, dimeric phases exist in an equilibrium between random coil, alpha-helical, beta-sheet, and beta-hairpin states. A thermodynamic characterization of the dimeric phases reveals that electric dipole-dipole interactions and optimal side-chain packing stabilize alpha-helical conformations, while hydrogen bond interactions favor beta-sheet conformations. Possible pathways leading to the formation of alpha-helical and beta-sheet dimers are discussed.

Ionic solvation studied by image-charge reaction field method

In a preceding paper [J. Chem. Phys. 131, 154103 (2009)], we introduced a new, hybrid explicit/implicit method to treat electrostatic interactions in computer simulations, and tested its performance for liquid water. In this paper, we report further tests of this method, termed the image-charge solvation model (ICSM), in simulations of ions solvated in water. We find that our model can faithfully reproduce known solvation properties of sodium and chloride ions. The charging free energy of a single sodium ion is in excellent agreement with the estimates by other electrostatics methods, while offering much lower finite-size errors. Similarly, the potentials of mean force computed for Na-Cl, Na-Na, and Cl-Cl pairs closely reproduce those reported previously. Collectively, our results demonstrate the superior accuracy of the proposed ICSM method for simulations of mixed media.