Bulletin of the American Physical Society
APS March Meeting 2013
Volume 58, Number 1
Monday–Friday, March 18–22, 2013; Baltimore, Maryland
Session M43: Focus Session: Protein Misfolding and Aggregation II |
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Sponsoring Units: DCP DBIO Chair: Joan Shea, UCSB Room: Hilton Baltimore Holiday Ballroom 2 |
Wednesday, March 20, 2013 8:00AM - 8:12AM |
M43.00001: In-Vivo Like Studies of the hIAPP Amyloid Precursors Using Dielectric Relaxation Spectroscopy Yusuke Hirai, Reem Mahommed Assiri, Donal Barry, Florin Despa, Izabela Stroe Recent studies show that the amyloid formation in Type II diabetic disease involves aggregation of monomers of the human islet amyloid polypeptide (hIAPP) into oligomers, protofibrils, and fibrils. Here we present data showing that Dielectric Relaxation Spectroscopy is a very sensitive technique to detect the hIAPP precursors. We measured the dielectric response of amyloidogenic hIAPP and non-amyloidogenic rIAPP as a function of frequency (10$^{-3}$ Hz to 10$^{7}$ Hz), temperature (193K to 283K), and incubation time (0-120 h). To mimic in-vivo like conditions, the proteins were measured in bovine serum albumin. Our results show that the dielectric signal of amyloidogenic hIAPP shifts towards the dielectric signal of the background, as predicted by theoretical calculations. No similar shift is observed for the non-amyloidogenic rIAPP. In addition, the dielectric signal of both the hIAPP and the rIAPP shows two relaxation processes over the measured temperature range. We used two Havrilik-Negami functions plus conductivity to fit the two relaxation processes we determined the relaxation time for both processes and calculated the corresponding activation energies. [Preview Abstract] |
Wednesday, March 20, 2013 8:12AM - 8:24AM |
M43.00002: Structure and Thermodynamic Stability of Islet Amyloid Polypeptide Monomers and Small Aggregates Chi-cheng Chiu, Sadanand Singh, Juan de Pablo Human islet amyloid polypeptide (hIAPP, also known as human amylin) is associated with the development of type II diabetes. It is known to form amyloid fibrils that are found in pancreatic islets. Pramlintide, a synthetic analog of hIAPP with three proline substitutions, is not amyloidogenic and has been applied in amylin replacement treatments. In this work, we use molecular simulations with advanced sampling techniques to examine the effect of these proline substitutions on hIAPP monomer conformations. We find that all three proline substitutions are required to attenuate the formation of $\beta $-sheets encountered in amylin. Furthermore, we investigate the formation of hIAPP dimers and trimers, and investigate how that process is affected by the presence of various additives. Our simulations show that hIAPP can form a $\beta $-sheet at the N-terminus and the C-terminus independently, in agreement with experimental observations. Our results provide valuable insights into the mechanism of hIAPP early aggregation and the design of fibril formation inhibitors. [Preview Abstract] |
Wednesday, March 20, 2013 8:24AM - 8:36AM |
M43.00003: Control the aggregation of model amyloid insulin protein under ac-electric fields Zhongli Zheng, Benxin Jing, Y. Elaine Zhu In vitro experiments have been widely used to characterize the misfolding/unfolding pathway characteristic of amylodogenic proteins. Conversion from natively folded amyloidogenic proteins to oligomers via nucleation is the accepted path to fibril formation upon heating over a certain lag time period. In an alternative engineering approach to manipulate and control protein aggregation, we have investigated the aggregation kinetics of insulin, a well-established amyloid model protein, under applied ac-electric fields of varied ac-frequency and voltage at room temperature. Using fluorescence correlation spectroscopy and fluorescence imaging, we have observed that the insulin aggregation can occur at much shortened lag time under applied ac-electric fields, when a critical ac-voltage is exceeded. The strong dependence of lag time on ac-frequency over a narrow range of 500 Hz-5 kHz indicates the effect of ac-electroosmosis on the diffusion controlled process of insulin nucleation. Yet, no difference of conformational structure is detected with insulin under applied ac-fields, suggesting the equivalence of ac-polarization to the conventional thermal activation process for insulin aggregation. [Preview Abstract] |
Wednesday, March 20, 2013 8:36AM - 9:12AM |
M43.00004: A physical chemical approach to understanding cellular dysfunction in type II diabetes Invited Speaker: Andrew Miranker The conversion of soluble protein into b-sheet rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g. Ab from Alzheimer's disease) reside in close association with a biological membranes. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the b-sheet rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically b-helical, or transiently adopt an a-helical state upon association with membrane. We have investigated these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. We report here the discovery of an oligomeric species that arises through stochastic nucleation on membranes, and results in disruption of the lipid bilayer. These species are stable, result in all-or-none leakage, and represent a definable protein/lipid phase that equilibrates over time. To characterize the reaction pathway of assembly, we apply an experimental design that includes ensemble and single particle evaluations \textit{in vitro} and correlate these with quantitative measures of cellular toxicity. [Preview Abstract] |
Wednesday, March 20, 2013 9:12AM - 9:48AM |
M43.00005: Amyloid Aggregation and Membrane Disruption by Amyloid Proteins Invited Speaker: Ayyalusamy Ramamoorthy Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.\\[4pt] References:\\[0pt] [1] Sciacca et al., \textit{Biophys. J.} 2012, \textbf{103}, 702-10.\\[0pt] [2] Sciacca et al., \textit{Biochemistry}. 2012, \textbf{51}, 7676-84\\[0pt] [3] Brender et al., \textit{Acc. Chem. Res.} 2012, \textbf{45}, 454-62.\\[0pt] [4] Nanga et al., \textit{Biochim. Biophys. Acta} 2011, \textbf{1808}, 2337-42.\\[0pt] [5] Brender et al., \textit{Biophys J.} 2011, \textbf{100}, 685-92. [Preview Abstract] |
Wednesday, March 20, 2013 9:48AM - 10:24AM |
M43.00006: New technology for 2D IR spectroscopy and its application to protein aggregation and drug binding Invited Speaker: Martin Zanni We are using 2D IR spectroscopy to study the aggregation and drug inhibition of proteins involved in common human diseases. It is extremely difficult to obtain precise structural information about drug inhibition of amyloid fibrillization, because it is very difficult to apply NMR spectroscopy and x-ray crystallography to these systems. As a result, there are very few molecular level details known about even the simplest inhibitors. We have studied a peptide inhibitor whose sequence was used to design an FDA approved drug, partially because this peptide has never before been observed to aggregate on its own. According to the sequence, we would expect that the C-terminal is responsible for inhibition, but in fact we found that the N-terminal was instead. In fact, we also observed that the complex formed between the inhibitor and amylin caused the inhibitor itself to form amyloid fibers. These surprising results were not previously observed, in part because the prior methods used to study inhibition was not sensitive to the specific structural fold of the fibers. [Preview Abstract] |
Wednesday, March 20, 2013 10:24AM - 10:36AM |
M43.00007: Early-Stage Aggregation of Islet Amyloid Polypeptide on Membrane Surfaces Probed by Label-Free Chiral Sum Frequency Generation Spectroscopy Zhuguang Wang, Li Fu, Elsa Yan The aggregation of human islet amyloid polypeptide (hIAPP) into fibrils is associated with type II diabetes. It can be catalyzed by interactions with membranes. Recent studies have shown that cytotoxicity arises from the intermediates of aggregation instead of mature fibrils. However, the pathogenic mechanism is still unknown and it remains challenging to probe structures of the intermediates on membrane surfaces due to a lack of biophysical methods that are sensitive to both protein secondary structures and interfaces. Here, we used label-free chiral sum frequency generation spectroscopy (cSFG) to probe the intermediates. Recently, we have discovered cSFG provides highly specific peptide vibrational signatures that can distinguish protein secondary structures at interfaces. Using cSFG, we observed in situ and in real time the aggregation of hIAPP from disordered structures to $\alpha $-helices and then $\beta $-sheets on membrane surfaces. We also obtained the orientation of the $\beta $-sheet aggregates inserted into the membranes. We further studied the S20G mutant, which is linked to the early onset of type II diabetes among Asian populations. We compared the mutant with the wild-type hIAPP to evaluate the effect of S20G in the early-stage aggregation on membrane surfaces. [Preview Abstract] |
Wednesday, March 20, 2013 10:36AM - 10:48AM |
M43.00008: Achiral and Chiral Sum Frequency Generation Spectroscopy of Peptides Joshua Carr, Lu Wang, James Skinner In vibrational sum-frequency generation (SFG) spectroscopy, a resonant IR and a non-resonant visible laser pulse are applied to a sample, and a signal is detected at the sum frequency of the pulses. This signal is sensitive to the local environments of interfacial chromophores. For the \emph{psp} polarization combination (\emph{p}-polarized SF, \emph{s}-polarized visible, \emph{p}-polarized IR), the signal is selectively sensitive to chiral structures. Recently, it was found that peptide secondary structures could be distinguished by the presence of absence of \emph{psp} signals for the amide I and NH stretch modes. This finding has been exploited to track the aggregation of human islet amyloid polypeptide at a water/air interface. To facilitate the interpretation of these experiments in terms of detailed structures, we present here a mixed quantum/classical method for the computation of both achiral and chiral SFG spectra for the peptide amide I mode, based on classical molecular dynamics simulations. We then apply this method to model systems, and comment as to the importance of both intrinsic chirality (the presence of atomic chiral centers) and structural chirality (the presence of chiral secondary structure) to the strength of the \emph{psp} signal. [Preview Abstract] |
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