Biomedical subjects

Thomas Wyttenbach

Publications and source records attributed to Thomas Wyttenbach.

11 recordsLinked to original sources

PubMed2007

Intermolecular interactions in biomolecular systems examined by mass spectrometry.

With the development of electrospray and matrix-assisted laser desorption ionization, mass spectrometry (MS) evolved into a powerful tool in the field of biochemistry. Whereas MS is primarily analytical in nature, an increasing number of MS research groups employ the method to address fundamental biochemical questions. Probing the interaction of noncovalently bound molecules in the mass spectrometer is one of the most interesting MS-based experiments possible today, with the potential of making a significant contribution to the basic understanding of the structure and function of biochemical complexes. Here we review a number of current research efforts employing primarily MS techniques to investigate intermolecular interactions in biochemical systems. Examples chosen include the interaction of biomolecules with solvent molecules; interactions between nucleic-acid molecules, in particular, interactions in duplex and quadruplex structures; and interactions between proteins involved in neurodegenerative diseases. Finally we conclude by presenting a few examples of very large biomolecular assemblies in the mega-Dalton range analyzed by MS.

Mass Spectrometry↗

PubMed2006

Hydration of mononucleotides.

The sequential addition of water molecules to protonated and deprotonated forms of the four mononucleotides dAMP, dCMP, dGMP, and dTMP was studied experimentally by equilibrium measurements using an electrospray mass spectrometer equipped with a drift cell and theoretically by computational methods including molecular modeling and density functional theory calculations. Experiments were carried out in positive and negative ion mode, and calculations included the protonated and deprotonated forms of the four nucleotides. For deprotonated anionic nucleotides the experimental enthalpies of hydration (DeltaH degrees n) were found to be similar for all four systems and varied between -10.1 and -11.5 kcal mol-1 for the first water molecule (n = 1) and -8.3 and -9.6 kcal mol-1 for additional water molecules (n = 2-4). Theory indicated that the first water molecule binds to the charge-carrying phosphate group. Simulations of deprotonated mononucleotides with four water molecules yielded a large number of structures with similar energies. In some of the structures all four water molecules cluster around the phosphate group, and in other structures the four water molecules each hydrate a different functional group of the nucleotide. These include the phosphate group, the deoxyribose hydroxyl group, and various functional groups on the nucleobases. Experimental DeltaH degrees 1 values for the protonated cationic mononucleotides ranged from -10.5 to -13.5 kcal mol-1 with more negative values (< or =-12 kcal mol-1) for dCMP, dGMP, and dTMP and the least negative value for dAMP. For n = 2-4 DeltaH degrees n values varied from -6.9 to -9.7 kcal/mol and were similar in value to the deprotonated nucleotides except for dAMP. Theory on the protonated nucleotides indicated that the first water molecule binds to the charge-carrying group for dCMP, dGMP, and dTMP. For protonated dAMP, on the other hand, the charge-carrying N3 group is well self-solvated by the phosphate group and not readily available for a hydrogen bond with the water molecule. The insight gained on nucleotide stabilization by individual water molecules is used to discuss the competition between hydration of individual nucleotides and Watson-Crick base pairing.

DNA↗

PubMed2006

Elucidating amyloid beta-protein folding and assembly: A multidisciplinary approach.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.

Amino Acid Sequence↗

PubMed2006

Amyloid beta-protein monomer structure: a computational and experimental study.

The structural properties of the Abeta42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's 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 Abeta42 peptide conformations are dominated by loops and turns but show some helical structure in the C-terminal hydrophobic tail. A second calculation on Abeta42 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 Abeta42 -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 Abeta42 are discussed.

Amyloid beta-Peptides↗

PubMed2006

Structure of the 21-30 fragment of amyloid beta-protein.

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(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.

Amyloid beta-Peptides↗

PubMed2005

Amyloid beta-protein: monomer structure and early aggregation states of Abeta42 and its Pro19 alloform.

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].

Amino Acid Sequence↗

PubMed2005

Oxytocin-receptor binding: why divalent metals are essential.

Biologists have observed that the presence of divalent metal is essential for the binding of the hormone oxytocin (OT) to its cellular receptor. However, this interaction is not understood on the molecular level. Because conformation is a key factor controlling ligand binding in biomolecule systems, we have used ion mobility experiments and molecular modeling to probe the conformation of the oxytocin-zinc complex. Results show that Zn2+ occupies an octahedral site in the interior of the OT peptide that frees the N-terminus and creates a structured hydrophobic binding site on the peptide exterior; both factors are conducive to binding oxytocin to its receptor.

Cations, Divalent↗

PubMed2004

Investigation of noncovalent interactions in deprotonated peptides: structural and energetic competition between aggregation and hydration.

Noncovalent peptide-peptide and peptide-water interactions in small model systems were examined using an electrospray mass spectrometer equipped with a high-pressure drift cell. The results of these aggregation and hydration experiments were interpreted with the aid of molecular mechanics (MM) and density functional theory (DFT) calculations. The systems investigated include bare deprotonated monomers and dimers [P(1,2)-H](-) and hydrated deprotonated monomers and dimers [P(1,2)-H](-).(H(2)O)(n)() for the peptides dialanine (P = AA) and diglycine (P = GG). Mass spectra indicated that both peptides AA and GG form exclusively dimer ions in the electrospray process. Monomeric ions were generated by high-energy injection of the dimers into the drift cell. Temperature-dependent hydration equilibrium experiments carried out in the drift cell yielded water binding energies ranging from 11.7 (first water molecule) to 7.1 kcal/mol (fourth water) for [AA-H](-) and 11.0 to 7.4 kcal/mol for [GG-H](-). The first water molecule adding to the dimer ions [AA-H](-).(AA) and [GG-H](-).(GG) is bound by 8.4 and 7.5 kcal/mol, respectively. The hydration mass spectra for the monomers and dimers provide a means to compare the ability of water and a neutral peptide to solvate a deprotonated peptide [P-H](-). The data indicate that a similar degree of solvation is achieved by four water molecules, [P-H](-).(H(2)O)(4), or one neutral peptide, [P-H](-).(P). Temperature-dependent kinetics experiments yielded activation energies for dissociation of the dimers [AA-H](-).(AA) and [GG-H](-).(GG) of 34.9 and 32.2 kcal/mol, respectively. MM and DFT calculations carried out for the dialanine system indicated that the dimer binding energy is 24.3 kcal/mol, when the [AA-H](-) and AA products are relaxed to their global minimum structures. However, a value of 38.9 kcal/mol is obtained if [AA-H](-) and AA dissociate but retain the structures of the moieties in the dimer, suggesting the transition state occurs early in the dissociation process. Similar results were found for the diglycine dimer.

Dipeptides↗

PubMed2004

Alpha-synuclein: stable compact and extended monomeric structures and pH dependence of dimer formation.

The protein alpha-synuclein, implicated in Parkinson's disease, was studied by combining nano-electrospray ionization (N-ESI) mass spectrometry and ion mobility. It was found that both the charge-state distribution in the mass spectra and the average protein shape deduced from ion mobility data, depend on the pH of the spray solution. Negative-ion N-ESI of pH 7 solutions yielded a broad charge-state distribution from -6 to -16, centered at -11, and ion mobility data consistent with extended protein structures. Data obtained for pH 2.5 solutions, on the other hand, showed a narrow charge-state distribution from -6 to -11, centered at -8, and ion mobilities in agreement with compact alpha-synuclein structures. The data indicated that there are two distinct families of structures: one consisting of relatively compact proteins with eight or less negative charges and one consisting of relatively extended structures with nine or more charges. The average cross section of a-synuclein at pH 2.5 is 33% smaller than for the extended protein sprayed from pH 7 solution. Significant dimer formation was observed when sprayed from pH 7 solution but no dimers were observed from the low pH solution. A plausible mechanism for aggregate formation in solution is proposed.

Amino Acid Sequence↗

PubMed2003

The effect of the initial water of hydration on the energetics, structures, and H/D exchange mechanism of a family of pentapeptides: an experimental and theoretical study.

A series of gas-phase experiments and extensive theoretical modeling was done on the family of singly protonated peptides AARAA, Ac-AARAA, and AARAA-OMe. (AARAA)H(+) underwent extensive H/D exchange with D(2)O, whereas the other two peptides with blocked termini did not, implying that a salt bridge was involved in the H/D exchange process. Ion mobility measurements and complementary molecular modeling unambiguously identified the 300 K structures of all three protonated peptides as charge solvation structures, not salt bridges. High-level density functional theory calculations indicated the global minimum of (AARAA)H(+) was a charge solvation structure with the lowest-energy salt bridge structure 4.8 kcal/mol higher in energy. Uptake of the first five water molecules of hydration at 260 K showed near identical propensities for all three peptides consistent with a common structural motif. Quantitative measurements of Delta H degrees and Delta S degrees for the first two waters of hydration were very similar for all three peptides, again suggestive of a common structure. A detailed search of the potential energy surface for the singly hydrated (AARAA)H(+) using molecular mechanics and density functional theory approaches indicated a charge solvation structure was the global minimum, but now the lowest-energy salt bridge structure was only 1.8 kcal/mol higher in energy. Importantly, a low-energy transition state connecting the charge solvation and the salt bridge structures was found where the D(2)O molecule facilitated H/D exchange via the relay mechanism. This "relay" transition state was 7 kcal/mol below the (AARAA)H(+) + D(2)O asymptotic energy, suggesting that facile H/D exchange could occur in this system. There was no equivalent low-lying relay mechanism transition state for the (Ac-AARAA)H(+) and (AARAA-OMe)H(+) peptides, consistent with the fact that H/D exchange was not observed. Hence, the combined experimental and theoretical methods confirmed that a salt bridge was involved in the H/D exchange by D(2)O of (AARAA)H(+), but it existed only as a kinetic intermediate, not as a global minimum structure. These findings suggest that caution must be observed in drawing structural conclusions from H/D exchange only. A prescription is given here for understanding both the structural and H/D exchange mechanistic aspects of bare and singly hydrated peptides.

Deuterium Exchange Measurement↗

PubMed2003

Sequential hydration of small protonated peptides.

The sequential addition of water molecules to a series of small protonated peptides was studied by equilibrium experiments using electrospray ionization combined with drift cell techniques. The experimental data were compared to theoretical structures of selected hydrated species obtained by molecular mechanics simulations. The sequential water binding energies were measured to be of the order of 7-15 kcal/mol, with the largest values for the first water molecule adding to either a small nonarginine containing peptide (e.g., protonated dialanine) or to a larger peptide in a high charge state (e.g., triply protonated neurotensin). General trends are (a) that the first water molecules are more strongly bound than the following water molecules, (b) that very small peptides (2-3 residues) bind the first few water molecules more strongly than larger peptides, (c) that the first few water molecules bind more strongly to higher charge states than to lower charge states, and (d) that water binds less strongly to a protonated guanidino group (arginine containing peptides) than to a protonated amino group. Experimental differential entropies of hydration were found to be of the order of -20 cal/mol/K although values vary from system to system. At constant experimental conditions the number of water molecules adding to any peptide ion is strongly dependent on the peptide charge state (with higher charge states adding proportionally more water molecules) and only weakly dependent on the choice of peptide. For small peptides molecular mechanics calculations indicate that the first few water molecules add preferentially to the site of protonation until a complete solvation shell is formed around the charge. Subsequent water molecules add either to water molecules of the first solvation shell or add to charge remote functional groups of the peptide. In larger peptides, charge remote sites generally compete more effectively with charge proximate sites even for the first few water molecules.

Arginine↗