Untersuchung der Druckantwort der random coil Modellpeptide Ac-GGXA-NH2 und der amyloidbildenden Polypeptide Aβ und IAPP mit Hilfe der Hochdruck-NMR-Spektroskopie

Die Hochdruck-NMR-Spektroskopie ist eine vielseitig einsetzbare Methode, mit der es möglich ist druckinduzierte Änderungen von Molekülen auf atomarer Ebene zu untersuchen. Die Anwendung von Druck erlaubt dabei die Verschiebung von Gleichgewichten und somit die Manipulation unterschiedlichster biologischer Interaktionen beziehungsweise die Verschiebung von Populationsgleichgewichten. Für die Hochdruck-NMR-Spektroskopie wurde ein Druckautoklav mit Keramikmesszelle entwickelt, in den zusätzliche Sicherheitsmerkmale integriert wurden, um im Falle des Bruchs der Keramik die Schäden am NMR-Spektrometer zu minimieren. Zudem wurden Druckeffekte an den amyloidogenen Polypeptiden Aβ (β-Amyloid) und IAPP (Insel-Amyloid-Polypeptid) untersucht und charakterisiert. Diese spielen eine wichtige Rolle bei der Alzheimer-Krankheit (Abeta) und Diabetes mellitus (IAPP). Dabei wurde gezeigt, dass es mit Hilfe der Hochdruck-NMR-Spektroskopie möglich ist verschiedene Zustände des β-Amyloids nachzuweisen. Des Weiteren wurde die druckinduzierte Depolymerisierung des β-Amyloids für unterschiedliche Temperaturen und Peptidkonzentration untersucht. Die so erhaltene Abhängigkeit der Monomerkonzentration vom Druck wurde unter Verwendung verschiedener Modelle thermodynamisch charakterisiert. Für das IAPP wurden die sequenzabhängigen Druckeffekte und Temperatureffekte des Peptidrückgrats bestimmt und mit strukturellen Eigenschaften des Peptids verknüpft. Um die strukturunabhängigen Druckeffekte der Aminosäuren zu bestimmen, wurde die Änderung der chemischen Verschiebung mit dem Druck der Aminosäure X3 im Modellpeptid Ac-GGXA-NH2 untersucht, wobei X für eine der 20 kanonischen Aminosäuren steht. Mit Hilfe der strukturunabhängigen Druckeffekte ist es möglich, Druckeffekte in Proteinen um den intrinsischen Druckeffekt der Aminosäuren zu korrigieren. Dabei erhält man die rein strukturabhängigen Druckeffekte des Proteins. Im Allgemeinen erfolgt die Beschreibung der druckinduzierten Änderung der chemischen Verschiebung mit einem polynominalen Modell zweiter Ordnung, da sich die Druckabhängigkeit in den meisten Fällen nicht linear verhält. Jedoch erhält man mit dieser Beschreibng keine Informationen über die thermodynamischen Parameter des Systems. In dieser Arbeit wurde gezeigt, dass sich das Koeffizientenverhältnis der polynominalen Koeffizienten mit thermodynamischen Parametern verknüpfen lässt

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes

NMR derived changes of lipoprotein particle concentrations related to impaired fasting glucose, impaired glucose tolerance, or manifest type 2 diabetes mellitus

Background Type 2 diabetes mellitus (T2D) and corresponding borderline states, impaired fasting glucose (IFG) and/or glucose tolerance (IGT), are associated with dyslipoproteinemia. It is important to distinguish between factors that cause T2D and that are the direct result of T2D. Methods The lipoprotein subclass patterns of blood donors with IFG, IGT, with IFG combined with IGT, and T2D are analyzed by nuclear magnetic resonance (NMR) spectroscopy. The development of lipoprotein patterns with time is investigated by using samples retained for an average period of 6 years. In total 595 blood donors are classified by oral glucose tolerance test (oGTT) and their glycosylated hemoglobin (HbA1c) concentrations. Concentrations of lipoprotein particles of 15 different subclasses are analyzed in the 10,921 NMR spectra recorded under fasting and non-fasting conditions. The subjects are assumed healthy according to the strict regulations for blood donors before performing the oGTT. Results Under fasting conditions manifest T2D exhibits a significant concentration increase of the smallest HDL particles (HDL A) combined with a decrease in all other HDL subclasses. In contrast to other studies reviewed in this paper, a general concentration decrease of all LDL particles is observed that is most prominent for the smallest LDL particles (LDL A). Under normal nutritional conditions a large, significant increase of the concentrations of VLDL and chylomicrons is observed for all groups with IFG and/or IGT and most prominently for manifest T2D. As we show it is possible to obtain an estimate of the concentrations of the apolipoproteins Apo-A1, Apo-B100, and Apo-B48 from the NMR data. In the actual study cohort, under fasting conditions the concentrations of the lipoproteins are not increased significantly in T2D, under non-fasting conditions only Apo-B48 increases significantly. Conclusion In contrast to other studies, in our cohort of “healthy” blood donors the T2D associated dyslipoproteinemia does not change the total concentrations of the lipoprotein particles produced in the liver under fasting and non-fasting conditions significantly but only their subclass distributions. Compared to the control group, under non-fasting conditions participants with IGT and IFG or T2D show a substantial increase of plasma concentrations of those lipoproteins that are produced in the intestinal tract. The intestinal insulin resistance becomes strongly observable

Ceramic cells for high pressure NMR spectroscopy of proteins

Application of high pressure to biological macromolecules can be used to find new structural states with a smaller specific volume of the system. High pressure NMR spectroscopy is a most promising analytical tool for the study of these states at atomic resolution. High pressure quartz cells are difficult to handle, high quality sapphire high pressure cells are difficult to obtain commercially. In this work, we describe the use of high pressure ceramic cells produced from yttrium stabilized ZrO2 that are capable of resisting pressures up to 200 MPa. Since the new cells should also be usable in the easily damageable cryoprobes a completely new autoclave for these cells has been constructed, including an improved method for pressure transmission, an integrated safety jacket, a displacement body, and a fast self-closing emergency valve.DF

Pulsed pressure perturbations, an extra dimension in NMR spectroscopy of proteins

The introduction of multidimensional NMR spectroscopy was a breakthrough in biological NMR methodology because it allowed the unequivocal correlation of different spin states of the system. The introduction of large pressure perturbations in the corresponding radio frequency (RF) pulse sequences adds an extra structural dimension into these experiments. We have developed a microprocessor-controlled pressure jump unit that is able to introduce fast, strong pressure changes at any point in the pulse sequences. Repetitive pressure changes of 80 MPa in the sample tube are thus feasible in less than 30 ms. Two general forms of these experiments are proposed here, the pressure perturbation transient state spectroscopy (PPTSS) and the pressure perturbation state correlation spectroscopy (PPSCS). PPTSS can be used to measure the rate constants and the activation energies and activation volumes for the transition between different conformational states including the folded and unfolded state of proteins, for polymerization-depolymerization processes, and for ligand binding at atomic resolution. PPSCS spectroscopy correlates the NMR parameters of different pressure-induced states of the system, thus allowing the measurement of properties of a given pressure induced state such as a folding intermediate in a different state, for example, the folded state. Selected examples for PPTSS and PPSCS spectroscopy are presented in this Article.DFGBFSFC

The pressure and temperature perturbation approach reveals a whole variety of conformational substates of amyloidogenic hIAPP monitored by 2D NMR spectroscopy

The intrinsically disordered human islet amyloid polypeptide (hIAPP) is a 37 amino acid peptide hormone that is secreted by pancreatic beta cells along with glucagon and insulin. The glucose metabolism of humans is regulated by a balanced ratio of insulin and hIAPP. The disturbance of this balance can result in the development of type-2 diabetes mellitus (T2DM), whose pathogeny is associated by self-assembly induced aggregation and amyloid deposits of hIAPP into nanofibrils. Here, we report pressure- and temperature-induced changes of NMR chemical shifts of monomeric hIAPP in bulk solution to elucidate the contribution of conformational substates in a residue-specific manner in their role as molecular determinants for the initial self-assembly. The comparison with a similar peptide, the Alzheimer peptide A beta(1-40), which is leading to self-assembly induced aggregation and amyloid deposits as well, reveals that in both peptides highly homologous areas exist (Q10-L16 and N21-L27 in hIAPP and Q15-A21 and S26-I32 in A beta). The N-terminal area of hIAPP around amino acid residues 3-20 displays large differences in pressure sensitivity compared to A beta, pinpointing to a different structural ensemble in this sequence element which is of helical origin in hIAPP. Knowledge of the structural nature of the highly amyloidogenic hIAPP and the differences with respect to the conformational ensemble of A beta(1-40) will help to identify molecular determinants of self-assembly as well as cross-seeded assembly initiated aggregation and help facilitate the rational design of drugs for therapeutic use

Conformational Substates of Amyloidogenic hIAPP Revealed by High Pressure NMR Spectroscopy

The islet amyloid polypeptide (IAPP) is a peptide hormone that is secreted by pancreatic beta cells along with glucagon and insulin. The development of type-2 diabetes mellitus (T2DM) is accompanied by aggregation and amyloid deposits of IAPP. Here we report pressure-induced changes of NMR chemical shifts for the elucidation of conformational substates of IAPP in bulk solution. Comparison with a similar peptide, the Alzheimer peptide Ab, reveals that the area around amino acid residues 3-20 displays large differences in the first and second order pressure coefficients, pinpointing to a different structural ensemble in this sequence element, while the area around amino acid residues 28-37 displays similar transient structural conformations for both peptides. Knowledge of the structural nature of the highly amyloidogenic IAPP and the differences with respect to the conformational ensemble of Ab will help facilitate the rational design of drugs for therapeutic treatment of T2DM

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of(1)H and(15)N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2(Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficientsB(1)andB(2)that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the(1)H and(15)N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For H(alpha)and H(N)correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients

Distinct conformational states of the Alzheimer β-amyloid peptide can be detected by high-pressure NMR spectroscopy

Folding under pressure: High-pressure NMR spectroscopy detects three different conformational states of the Aβ-peptide in solution: a compactly folded state 1, a partially folded state 2′, and a random-coil like state 2′′ (see plot, p=population). At ambient pressure the folded state 1 dominates which probably has a high affinity to fibrils and thus may promote fibril formation.DFGBayerische Forschungsstiftung (BFS)Fonds der Chemischen Industrie (FCI)FAPES

Pressure dependence of backbone chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms H-1(alpha), C-13(alpha) and C-13' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The polynomial pressure coefficients B (1) and B (2) are dependent on the type of amino acid studied. The coefficients of a given nucleus show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure are also weakly correlated