From protons to OXPHOS supercomplexes and Alzheimer's disease: Structure–dynamics–function relationships of energy-transducing membranes

AbstractBy the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I1III2IV1, in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimer's dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies

EFFECT OF HYDRATION AND MACROMOLECULAR CROWDING ON PEPTIDE CONFORMATION, AGGREGATION AND FOLDING KINETICS

Protein folding/misfolding in vivo takes place in a highly crowded and confined environment. Such crowded environment can possibly lead to fewer water molecules surrounding a protein of interest than that seen under in vitro conditions wherein typically dilute aqueous solutions are used. When considering the aforesaid cellular characteristics, such as water depletion and macromolecular crowding; it is reasonable to assume that proteins may experience different energy landscapes when folding in vivo than in vitro. Therefore, we have investigated how degrees of hydration and macromolecular crowding affect the conformation, aggregation and folding kinetics of short peptides. In order to modulate the number of water molecules accessible to the peptide molecules of interest, we encapsulated the peptides in the aqueous core of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and isooctane (IO) at different water loadings. Using this reverse micellar platform, we systematically studied the conformation and aggregation properties of alanine-based peptides and amyloid forming segments derived from amyloid beta peptides and yeast prion protein Sup35 at different hydration levels. Our studies demonstrated that limited hydration facilitates aggregate formation in these peptides and that removal of water imposes a free energy barrier to peptide association and aggregation. These studies have implications for understanding aggregate/amyloid formation in vivo where macromolecular crowding can change the solvation status of the peptides. Furthermore, we examined how the folding dynamics of secondary/supersecondary structural elements are modulated by a crowded environment in comparison to that of dilute aqueous solutions. To this effect we studied the thermal stability and folding-unfolding kinetics of three small folding motifs, i.e., a 34-residue alpha-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue beta-hairpin, in the presence of crowding agents (i.e. inert high mass polymers). Our results indicate that the folding-unfolding transition of alpha-helical peptides is insensitive to macromolecular crowding. However, we find that crowding leads to an appreciable decrease in the folding rate of the shortest beta-hairpin peptide. We propose a model considering both the static and dynamic effects arising from the presence of the crowding agent to rationalize these results

Amyloid Fibril Nucleation In Reverse Micelles

The 40-residue amyloid beta protein (Abeta) is the unstructured cleavage product of a common membrane protein that is produced in large quantities, but normally cleared from the brain before it exerts any apparent toxicity. Under some conditions, however, it undergoes a conformational change and aggregates into fibrils. These fibrils then coalesce into amyloid plaques, which are the pathognomonic brain lesions of Alzheimer‘s disease. The plaques are centers of active oxidative stress and neuronal death, so the conditions under which fibrils form is of high interest. When Abeta is encapsulated in a reverse micelle, its infrared spectrum indicates that it spontaneously adopts a fibril-like structure, which is remarkable because only one Abeta strand is present in each reverse micelle. That observation suggests that some aspect of the reverse micelle environment such as crowding, dehydration, proximity to a membrane, or high ionic strength may induce Abeta to nucleate amyloid fibril formation. Therefore, an understanding of the factors that induce Abeta to adopt fibril-like structure in reverse micelles may reveal what causes amyloid fibrils to form in Alzheimer\u27s disease. Molecular dynamics simulations of Abeta in reverse micelles have been performed to identify and understand these factors. Results indicate that Abeta side chains penetrate the reverse micelle surface, anchoring the peptide in the membrane. Other interactions between peptide and membrane stabilize intrachain hydrogen bond formation and secondary structure. These interactions may be important factors in the formation of amyloid fibrils and the pathogenesis of Alzheimer‘s disease

Solid State NMR Investigations of Lipid Bilayers and Biomembrane Binding Molecules: Dendrimers and Amylin.

The goal of this project was to investigate the interaction between lipid binding molecules, such as peptides and drug compounds, with biological lipid bilayers. The results demonstrate that human amylin, a peptide hormone implicated in type II adult onset diabetes, disrupts cell membranes via a curvature inducing mechanism. In order to assess the effects that the various membrane-associated molecules have on lipid bilayers, a combination of differential scanning calorimetry (DSC) and solid state NMR methods were used. The amyloidogenic and toxic hIAPP1-37 peptide, the non-toxic and non-amyloidogenic rIAPP1-37 peptide, and the toxic but largely non-amyloidogenic rIAPP1-19 and hIAPP1-19 fragments were characterized. It is also shown that hyperbranched polymers with nanotherapeutic applications, known as poly(amidoamine) dendrimers, are thermodynamically stable when inserted inside zwitterionic lipid bilayers using 1H radio frequency driven dipolar recoupling (RFDR) and 1H magic angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) techniques. 14N and 31P NMR experiments on static samples and measurements of the mobility of C-H bonds using a 2D proton detected local field protocol under MAS corroborate these results. The localization of dendrimers in the hydrophobic core of lipid bilayers restricts the motion of bilayer lipid tails, with the smaller G5 dendrimer having more of an effect than the larger G7 dendrimer. Furthermore, solid state NMR techniques are developed to study the lipid bilayers and the molecules that associate with them. These methods drastically increase the spectral resolution of 2D solid state NMR techniques and allow more accurate structure and dynamics information to be extracted from solid phase NMR samples. These techniques, known as separated local field (SLF) techniques, are used to obtain information about the orientation dependent local fields at each chemical site in a molecule under investigation. SLF techniques have been very important in the extraction of structural information from aligned samples in the solid state. Furthermore, a method is proposed to enable resonance assignment to be made on uniformly labeled samples. This method promises to overcome many of the difficulties inherent in solid state NMR studies of the structure and dynamics of biological membranes and the molecules that associate with them.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77927/1/smpieter_1.pd

Spectroscopic Probes of Protein Structure, Dynamics, Hydration and Electrostatics

The structure, dynamics and function of a protein are intimately controlled by a large number of intermolecular and intramolecular interactions. Thus, achieving a quantitative and molecular-level understanding of how proteins fold and function requires experimental techniques that can â??senseâ?? and differentiate various molecular forces and, in many cases, in a site-specific manner. To that end, the focus of this thesis work is to develop non-natural amino acid-based infrared and fluorescence probes that can be used to assess the local hydration status and electrostatic environment of proteins. First, we expand the utility of a well-known site-specific spectroscopic probe, p-cyano-phenylalanine (PheCN), by showing that (1) its fluorescence is sensitive to the presence of various anions and can thus be used to measure the heterogeneity of the protein conformation, (2) when placed at the N-terminal end of a peptide this non-natural amino acid can be used as a pH sensor for a wide variety of applications, and (3) its nitrile stretching vibration is a microscopic reporter of how a co-solvent, such as urea and trimethylamine N-oxide, modulates the protein-water interactions. Secondly, we demonstrate that the ester carbonyl stretching vibration of the non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, can be used to site-specifically quantify the electrostatic environment of proteins as its vibrational frequency correlates linearly with the local electrostatic field. Application of this infrared probe to amyloids allows us to gain new insight into their structure and dynamics

In-Vivo Like Studies of the hIAPP Amyloid Precursors by DRS

Recent studies show the amyloid formation in Type II diabetic disease involves aggregation of monomers of the human islet amyloid polypeptide (hIAPP) into oligomers, protofibrils, and fibrils. We present data show Dielectric Relaxation Spectroscopy is a sensitive technique to detect the hIAPP precursors. We measured the dielectric response of the hIAPP and the rIAPP as a function of frequency, temperature, and incubation time. In-vivo like conditions were mimiced by BSA. Our results show the dielectric signal of the hIAPP shifts towards the dielectric signal of the background while the rIAPP did not. The dielectric signal of the hIAPP and the rIAPP shows two relaxation processes over the measured range. We used two Havrilik-Negami functions and conductivity to fit the relaxation processes

Driving forces and structural determinants of steric zipper peptide oligomer formation elucidated by atomistic simulations.

Understanding the structural and energetic requirements of non-fibrillar oligomer formation harbors the potential to decipher an important yet still elusive part of amyloidogenic peptide and protein aggregation. Low-molecular-weight oligomers are described to be transient and polymorphic intermediates in the nucleated self-assembly process to highly ordered amyloid fibers and were additionally found to exhibit a profound cytotoxicity. However, detailed structural information on the oligomeric species involved in the nucleation cannot be readily inferred from experiments. Here, we study the spontaneous assembly of steric zipper peptides from the tau protein, insulin and α-synuclein with atomistic molecular dynamics simulations on the microsecond timescale. Detailed analysis of the forces driving the oligomerization reveals a common two-step process akin to a general condensation-ordering mechanism and thus provides a rational understanding of the molecular basis of peptide self-assembly. Our results suggest that the initial formation of partially ordered peptide oligomers is governed by the solvation free energy, whereas the dynamical ordering and emergence of β-sheets are mainly driven by optimized inter-peptide interactions in the collapsed state. A novel mapping technique based on collective coordinates is employed to highlight similarities and differences in the conformational ensemble of small oligomer structures. Elucidating the dynamical and polymorphic β-sheet oligomer conformations at atomistic detail furthermore suggests complementary sheet packing characteristics similar to steric zipper structures, but with a larger heterogeneity in the strand alignment pattern and sheet-to-sheet arrangements compared to the cross-β motif found in the fibrillar or crystalline states

Mass transport phenomena at the solid-liquid nanoscale interface in biomedical application

Understanding heat and mass transfer phenomena at solid-liquid nanoscale interface plays a crucial role for introducing novel and more rationally designed theranostic particles, drug with tailored features and for gaining new insight on the biomolecules functioning. For instance, the water transport properties in the proximity of Amyloid beta peptides can influence the formation of amyloid plaques found in the brains of Alzheimer patients. In the present work, transport behavior of water molecules in nanoconfined conditions has been investigated. By means of equilibrium Molecular Dynamics (MD) simulations, characteristic length of water confinement has been evaluated in the proximity of several biomolecules such as proteins and amino acids. Moving from proteins to their building blocks (i.e. amino acids), a similarity in water behavior was initially expected; MD simulations results show, instead, a more complex picture revealing a difference between the potential of water nanoconfinement by either proteins or amino acids. Hence, the reduction of water mobility in the proximity of nanoscale interfaces does not rely only on the local physical and chemical properties of the biomolecules surface, but the effects of size and potentials overlap should be also taken into account

NMR Investigations of the Self-Organization and Dynamics of Mutated Amyloid Protein Fibrils

This work investigates the influence of mutations at selected positions on the structure formation of the Alzheimer’s disease peptide amyloid β. Amyloid β is a member of the class of intrinsically disordered proteins that can aggregate into fibrils, which are characterized by a highly stable secondary structure, called cross-β structure. A central contact during fibrillation is the hydrophobic F19-L34 contact, which is located within the core of the cross-β structure. Modifications of this contact are known to influence the local molecular structure whereas the fibril morphology and the cross-β structure remain stable. In contrast, toxicity of amyloid β was completely lost for all previously investigated mutants of F19 and L34. This work characterizes the properties of this contact and answers the question what the minimally tolerated modifications are. To characterize the structure, structure formation process and biological activity of the Aβ variants a set of experiments was carried out. The local structure and dynamics were investigated using NMR experiments focusing on 13C-chemical shift changes and 1H-13C dipolar couplings, respectively. The fibril morphology and cross- β structure was verified by electron microscopy, circular dichroism spectroscopy and X-ray diffraction. Toxicity and biological activity was investigated using complementary cell culture experiments. The work was divided in three parts. First, L34 was substituted with three highly similar amino acids: the isomer isoleucine, valine that is one methylene group shorter but also a branched chain amino acid and the stereoisomer D-leucine. The L34 position proved to be important for the initiation of the structure formation, oligomer stability, fibril growth and the biological activity of amyloid β. These characteristics and properties were highly sensitive also to minor modifications but the different mutants showed no specific but qualitatively similar effects. The second part complemented previous mutation studies of the F19 position. Four new mutants were designed testing mild modification of the F19-L34 contact: phenylglycine and the homophenylalanine (S)-2-amino-4-phenyl-butyric acid change the length of the side chain, cyclohexyl-alanine eliminates the π-aromaticity of the ring system and increases the 3D steric demand, and (1-naphtyl)-alanine increases the 2D steric demand while maintaining the aromaticity. Mutations at the F19 position caused qualitatively similar effects as L34 modifications but proved to have quantitatively greater impact. Furthermore, they showed some specificity as steric constraints caused larger changes than modifications of the ring system. The third part investigates the influence of β-methylamino-L-alanine (BMAA) substitutions at positions F19, S8, and S26. The serine to BMAA substitutions were included because of their potential medical relevance. A F19BMAA substitution caused similar effects like other modifications at this position. Replacement of serine lead to a structural reorientation of the Aβ N-terminus and turn region. Furthermore, the pathways of the cell response changed from mitochondrial activity and plasma membrane integrity to apoptosis and neuronal stress reaction. Summarizing, it could be shown that, although the formation and structure of amyloid β fibrils is robust against different modifications the fibrillation kinetics, local structure and especially biological activity is highly sensitive and to some extend specific to even minor modifications

Solid-state NMR characterization of Alzheimer-like tau amyloid fibrils.

Eines der bedeutendsten Kennzeichen des Morbus Alzheimer ist die Zusammenlagerung des Mikrotubuli-assoziierten Proteins Tau in Fibrillen, die als „gepaarte helikale Filamente“ (PHF) bezeichnet werden. Allerdings sind die strukturellen Grundlagen der PHF Aggregation auf atomarer Ebene weitestgehend unbekannt. In dieser Studie wurden mittels Festkörper-Kernspinresonanz-Spektroskopie (FK-NMR) in vitro hergestellte PHF einer Tau Isoform untersucht, die aus drei Wiederholungseinheiten besteht und den Kern der PHF repräsentiert (K19). Wir haben herausgefunden, dass der rigide Kern der Fibrillen von den Aminosäuren V306 bis S324 – lediglich 18 von 99 Residuen – gebildet wird und aus 3 β-Faltblatt-Strängen besteht, die durch zwei kurze Knickstellen miteinander verbunden sind. Der erste β-Strang wird von dem gut untersuchten Hexapeptid 306VQIVYK311 gebildet. Von diesem ist bekannt, dass es sich ebenfalls zusammenlagern kann und dabei so genannte hydrophobe „steric zipper“ Kontakte ausbildet. Ergebnisse an einer gemischt [15N:13C]-markierten K19 PHF Probe zeigen, dass sich die β-Stränge parallel und nicht zu einander verschoben übereinander lagern. Zwischen C322-Resten verschiedener Moleküle bilden sich Disulfid-Brücken (DSB) aus, die zu einer lokalen Beeinträchtigung der β-Faltblatt-Struktur führen, wodurch in den FK-NMR Spektren Polymorphismus beobachtbar ist. Insbesondere die Aminosäurereste K321-S324 weisen zwei Resonanz-Sätze auf. Des Weiteren bestätigen Experimente, die an K19 C322A PHF durchgeführt wurden, den Einfluss der DSB auf die Struktur des Fibrillenkerns. Die Strukturdaten werden durch H/D-Austausch NMR Messungen an K19 sowie K18, einer Isoform bestehend aus vier Wiederholungseinheiten, gestützt. Zielgerichtete Mutagenese-Studien an K19 zeigen, dass Mutationen innerhalb der drei verschiedenen β-Stränge zu einem signifikanten Verlust der PHF Aggregationseffizienz führen, was die Bedeutung der β-Strang-reichen Region für die Zusammenlagerung von Tau Proteinen unterstreicht