Publications by authors named "Ayyalusamy Ramamoorthy"

277 Publications

Solid-state packing dictates the unexpected solubility of aromatic peptides.

Cell Rep Phys Sci 2021 Apr 21;2(4):100391. Epub 2021 Apr 21.

Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.

The understanding and prediction of the solubility of biomolecules, even of the simplest ones, reflect an open question and unmet need. Short aromatic tripeptides are among the most highly aggregative biomolecules. However, in marked contrast, Ala-Phe-Ala (AFA) was surprisingly found to be non-aggregative and could be solubilized at millimolar concentrations. Here, aiming to uncover the underlying molecular basis of its high solubility, we explore in detail the solubility, aggregation propensity, and atomic-level structure of the tripeptide. We demonstrate an unexpectedly high water solubility of AFA reaching 672 mM, two orders of magnitude higher than reported previously. The single crystal structure reveals an anti-parallel β sheet conformation devoid of any aromatic interactions. This study provides clear mechanistic insight into the structural basis of solubility and suggests a simple and feasible tool for its estimation, bearing implications for design of peptide drugs, peptides materials, and advancement of peptide nanotechnology.
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http://dx.doi.org/10.1016/j.xcrp.2021.100391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063180PMC
April 2021

Investigation of the effects of two major secretory granules components, insulin and zinc, on human-IAPP amyloid aggregation and membrane damage.

Chem Phys Lipids 2021 Apr 19;237:105083. Epub 2021 Apr 19.

Biophysics Program, Department of Chemistry, Biomedical Engineering, and Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the β-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.
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http://dx.doi.org/10.1016/j.chemphyslip.2021.105083DOI Listing
April 2021

Degradation of Alzheimer's amyloid-β by a catalytically inactive insulin-degrading enzyme.

J Mol Biol 2021 Apr 15:166993. Epub 2021 Apr 15.

Biophysics, Department of Chemistry, Macromolecular Engineering and Science, and Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.
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http://dx.doi.org/10.1016/j.jmb.2021.166993DOI Listing
April 2021

Benchmarks of SMA-Copolymer Derivatives and Nanodisc Integrity.

Langmuir 2021 03 1;37(10):3113-3121. Epub 2021 Mar 1.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States.

Poly(styrene--maleic acid) or SMA and its derivatives, a family of synthetic amphipathic copolymers, are increasingly used to directly solubilize cell membranes to functionally reconstitute membrane proteins in native-like copolymer-lipid nanodiscs. Although these copolymers act, de facto, like a "macromolecular detergent", the polymer-based lipid-nanodiscs has been demonstrated to be an excellent membrane mimetic for structural and functional studies of membrane proteins and their complexes by a variety of biophysical and biochemical approaches. In many studies reported in the literature, the choice of the right SMA formulation can depend on a number of factors, and the experimental conditions are typically developed according to a trial-and-error process since each studied system requires adapted protocols. While increasing number of nanodisc-forming copolymers are reported to be useful and they provide flexibilities in optimizing the sample preparation conditions, it is important to develop a systematic protocol that can be used for various applications. In this context, there is a vital necessity of benchmarking the performances of existing copolymer formulations, assessing crucial parameters for the successful extraction, isolation, and stabilization of membrane proteins. In this study, we compare both copolymers and copolymer-lipid nanodiscs obtained by SMA-EA with a set of anionic XIRAN copolymer formulations commercially available under the names of SL25010 P, SL30010 P, and SL40005 P. The reported results show how the critical micellar concentration (c.m.c.) of each copolymer is significantly altered in the presence of lipids and confirms the existence of an equilibrium between nanodisc-bound and "free" or "micellar" copolymer chains in the solution. We believe that these findings can be exploited to optimize studies that involve the necessity of special copolymers, which would not only simplify the applications but also broaden the scope of polymer-based nanodiscs.
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http://dx.doi.org/10.1021/acs.langmuir.0c03554DOI Listing
March 2021

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Chem Rev 2021 Feb 5;121(4):2545-2647. Epub 2021 Feb 5.

Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, , , and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.
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http://dx.doi.org/10.1021/acs.chemrev.0c01122DOI Listing
February 2021

Proteostasis of Islet Amyloid Polypeptide: A Molecular Perspective of Risk Factors and Protective Strategies for Type II Diabetes.

Chem Rev 2021 Feb 11;121(3):1845-1893. Epub 2021 Jan 11.

Biophysics, Department of Chemistry, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan 41809-1055, United States.

The possible link between hIAPP accumulation and β-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, β-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.
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http://dx.doi.org/10.1021/acs.chemrev.0c00981DOI Listing
February 2021

Lipid-Chaperone Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins.

ACS Chem Neurosci 2020 12 3;11(24):4336-4350. Epub 2020 Dec 3.

Department of Chemical Sciences, University of Catania, Catania 95124, Italy.

An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-β, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-β, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-β, and α-synuclein) and nonamyloidogenic (rat IAPP, β-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.
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http://dx.doi.org/10.1021/acschemneuro.0c00588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769131PMC
December 2020

Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology.

Biophys Chem 2021 Feb 19;269:106507. Epub 2020 Nov 19.

Biophysics, University of Michigan, Ann Arbor, MI 48109, USA; Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA; Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Abnormal aggregation of proteins into filamentous aggregates commonly associates with many diseases, such as Alzheimer's disease, Parkinson's disease and type-2 diabetes. These filamentous aggregates, also known as amyloids, can propagate their abnormal structures to either the same precursor molecules (seeding) or other protein monomers (cross-seeding). Cross-seeding has been implicated in the abnormal protein aggregation and has been found to facilitate the formation of physiological amyloids. It has risen to be an exciting area of research with a high volume of published reports. In this review article, we focus on the biophysical processes underlying the cross-seeding for some of the most commonly studied amyloid proteins. Here we will discuss the relevant literature related to cross-seeded polymerization of amyloid-beta, human islet amyloid polypeptide (hIAPP, or also known as amylin) and alpha-synuclein. SEVI (semen-derived enhancer of viral infection) amyloid formation by the cross-seeding between the bacterial curli protein and PAP is also briefly discussed.
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http://dx.doi.org/10.1016/j.bpc.2020.106507DOI Listing
February 2021

Small molecule induced toxic human-IAPP species characterized by NMR.

Chem Commun (Camb) 2020 Nov 2;56(86):13129-13132. Epub 2020 Oct 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.
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http://dx.doi.org/10.1039/d0cc04803hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641245PMC
November 2020

Natural-abundance O NMR spectroscopy of magnetically aligned lipid nanodiscs.

Chem Commun (Camb) 2020 Aug;56(69):9998-10001

Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.

Natural-abundance 17O NMR experiments are used to investigate the hydrated water in magnetically aligned synthetic polymer based lipid-nanodiscs. Residual quadrupole couplings (RQCs) measured from the observed five 17O (central and satellite) transitions, and molecular dynamics simulations, are used to probe the ordering of water molecules across the lipid bilayer.
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http://dx.doi.org/10.1039/d0cc04011hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484029PMC
August 2020

Amylin and beta amyloid proteins interact to form amorphous heterocomplexes with enhanced toxicity in neuronal cells.

Sci Rep 2020 06 25;10(1):10356. Epub 2020 Jun 25.

School of Pharmacy and Biomedical Sciences, Curtin Health and Innovation Research Institute (CHIRI), Faculty of Health Sciences, Curtin University, Bentley, WA, 6107, Australia.

Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aβ) can accumulate in Type 2 diabetes (T2D) and Alzheimer's disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aβ-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aβ oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aβ42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aβ and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aβ-mediated neuronal cell death. rIAPP exhibited reductions in Aβ induced neuronal cell death that was independent of its ability to interact with Aβ and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aβ aggregation and toxicity and provide new insight into the potential pathogenic effects of Aβ-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.
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http://dx.doi.org/10.1038/s41598-020-66602-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316712PMC
June 2020

High-Speed Atomic Force Microscopy Reveals the Structural Dynamics of the Amyloid-β and Amylin Aggregation Pathways.

Int J Mol Sci 2020 Jun 16;21(12). Epub 2020 Jun 16.

Division of Neurology, Department of Internal Medicine, School of Medicine, Showa University, Hatanodai, Shinagawa district, Tokyo 142-8666, Japan.

Individual Alzheimer's disease (AD) patients have been shown to have structurally distinct amyloid-β (Aβ) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aβ fibril structures. Thus, the characterization of the structural dynamics of Aβ could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aβ and amylin, which is associated with type II diabetes, shares some common biological features with Aβ, and is reported to be involved in AD.
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http://dx.doi.org/10.3390/ijms21124287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352471PMC
June 2020

High-Throughput Screening at the Membrane Interface Reveals Inhibitors of Amyloid-β.

Biochemistry 2020 06 5;59(24):2249-2258. Epub 2020 Jun 5.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.

Aggregation and the formation of oligomeric intermediates of amyloid-β (Aβ) at the membrane interface of neuronal cells are implicated in the cellular toxicity and pathology of Alzheimer's disease. Small molecule compounds have been shown to suppress amyloid aggregation and cellular toxicity, but often the presence of a lipid membrane negates their activity. A high-throughput screen of 1800 small molecules was performed to search for membrane active inhibitors, and 21 primary hits were discovered. Through the use of fluorescence-based assays, transmission electron microscopy, and dot blot assays, the initial 21 primary hits were narrowed down to five lead compounds. Nuclear magnetic resonance and circular dichroism experiments were used for further confirmation of amyloid inhibition at the membrane interface and to obtain insights into the secondary structure of amyloid-β, while size exclusion chromatography was used to characterize the size of Aβ species. Lastly, dye-leakage assays allowed us to understand how the addition of the five lead compounds affected amyloid-β's ability to permeate the lipid bilayer. These results provide insights into small molecules that stabilize small amyloid species in the presence of membranes for the development of tool compounds for deeper investigations of these transient species.
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http://dx.doi.org/10.1021/acs.biochem.0c00328DOI Listing
June 2020

Detergent-free extraction, reconstitution and characterization of membrane-anchored cytochrome-b5 in native lipids.

Chem Commun (Camb) 2020 Jun 28;56(48):6511-6514. Epub 2020 May 28.

Biophysics Program, Department of Chemistry, Macromolecular Science and Engineering, University of Michigan, Arbor, MI 48109-1055, USA.

Despite their denaturing properties, detergents are used to purify and study membrane proteins. Herein, we demonstrated a polymer-based detergent-free extraction of the membrane protein cytochrome-b5 along with E. coli lipids. Nuclear magnetic resonance experiments revealed the suitability of using nanodiscs for high-resolution studies and revealed the types of native lipids associated with the protein.
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http://dx.doi.org/10.1039/d0cc01737jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337974PMC
June 2020

Lipid-nanodiscs formed by paramagnetic metal chelated polymer for fast NMR data acquisition.

Biochim Biophys Acta Biomembr 2020 09 1;1862(9):183332. Epub 2020 May 1.

Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA; Biophysics and Chemistry Department, The University of Michigan, Ann Arbor, MI 48109-1055, USA; Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA; Biomedical Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

Lipid-nanodiscs have been shown to be an exciting innovation as a membrane-mimicking system for studies on membrane proteins by a variety of biophysical techniques, including NMR spectroscopy. Although NMR spectroscopy is unique in enabling the atomic-resolution investigation of dynamic structures of membrane-associated molecules, it, unfortunately, suffers from intrinsically low sensitivity. The long data acquisition often used to enhance the sensitivity is not desirable for sensitive membrane proteins. Instead, paramagnetic relaxation enhancement (PRE) has been used to reduce NMR data acquisition time or to reduce the amount of sample required to acquire an NMR spectra. However, the PRE approach involves the introduction of external paramagnetic probes in the system, which can induce undesired changes in the sample and on the observed NMR spectra. For example, the addition of paramagnetic ions, as frequently used, can denature the protein via direct interaction and also through sample heating. In this study, we show how the introduction of paramagnetic tags on the outer belt of polymer-nanodiscs can be used to speed-up data acquisition by significantly reducing the spin-lattice relaxation (T) times with minimum-to-no alteration of the spectral quality. Our results also demonstrate the feasibility of using different types of paramagnetic ions (Eu, Gd, Dy, Er, Yb) for NMR studies on lipid-nanodiscs. Experimental results characterizing the formation of lipid-nanodiscs by the metal-chelated polymer, and their increased tolerance toward metal ions are also reported.
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http://dx.doi.org/10.1016/j.bbamem.2020.183332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340147PMC
September 2020

High-resolution probing of early events in amyloid-β aggregation related to Alzheimer's disease.

Chem Commun (Camb) 2020 Apr 17;56(34):4627-4639. Epub 2020 Apr 17.

Biophysics Program, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.

In Alzheimer's disease (AD), soluble oligomers of amyloid-β (Aβ) are emerging as a crucial entity in driving disease progression as compared to insoluble amyloid deposits. The lacuna in establishing the structure to function relationship for Aβ oligomers prevents the development of an effective treatment for AD. While the transient and heterogeneous properties of Aβ oligomers impose many challenges for structural investigation, an effective use of a combination of NMR techniques has successfully identified and characterized them at atomic-resolution. Here, we review the successful utilization of solution and solid-state NMR techniques to probe the aggregation and structures of small and large oligomers of Aβ. Biophysical studies utilizing the commonly used solution and F based NMR experiments to identify the formation of small size early intermediates and to obtain their structures, and dock-lock mechanism of fiber growth at atomic-resolution are discussed. In addition, the use of proton-detected magic angle spinning (MAS) solid-state NMR experiments to obtain high-resolution insights into the aggregation pathways and structures of large oligomers and other aggregates is also presented. We expect these NMR based studies to be valuable for real-time monitoring of the depletion of monomers and the formation of toxic oligomers and high-order aggregates under a variety of conditions, and to solve the high-resolution structures of small and large size oligomers for most amyloid proteins, and therefore to develop inhibitors and drugs.
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http://dx.doi.org/10.1039/d0cc01551bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254607PMC
April 2020

High-resolution proton-detected MAS experiments on self-assembled diphenylalanine nanotubes enabled by fast MAS and high magnetic field.

J Magn Reson 2020 04 17;313:106717. Epub 2020 Mar 17.

Biophysics and Department of Chemistry, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

The advent of ultrahigh magnetic field and fast magic-angle-spinning (MAS) probe technology has led to dramatically enhanced spectral resolution and sensitivity in solid-state NMR spectroscopy. In particular, proton-based multidimensional solid-state NMR techniques have become feasible to investigate the structure and dynamics at atomic resolution, due to the increased chemical shift span and spectral resolution. Herein, the benefits of faster MAS and higher magnetic field are demonstrated on a self-assembled diphenylalanine (Phe-Phe) nanomaterial. Proton-detected 2D H/H single-quantum/single-quantum (SQ/SQ) correlation, double-quantum/single-quantum (DQ/SQ) correlation, and H chemical shift anisotropy/chemical shift (CSA/CS) correlation spectra obtained at two different spinning speeds (60 and 100 kHz) and two different magnetic fields (600 and 900 MHz) are reported. The dramatic enhancement of proton spectral resolution achieved with the use of a 900 MHz magnetic field and 100 kHz MAS is remarkable and enabled the measurement of proton CSA tensors, which will be useful to better understand the self-assembled structures of Phe-Phe nanotubes. We also show through numerical simulations that the unaveraged proton-proton dipolar couplings can result in broadening of CSA lines, leading to inaccurate determination of CSA tensors of protons. Thus, our results clearly show the insufficiency of a 600 MHz magnetic field to resolve H spectra lines and the inability of a moderate spinning speed of 60 kHz to completely suppress H-H dipolar couplings, which further justify the pursuit of ultrahigh magnetic field beyond 1 GHz and ultrafast MAS beyond 100 kHz.
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http://dx.doi.org/10.1016/j.jmr.2020.106717DOI Listing
April 2020

Symmetry-breaking transitions in the early steps of protein self-assembly.

Eur Biophys J 2020 Mar 2;49(2):175-191. Epub 2020 Mar 2.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly β-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aβ(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.
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http://dx.doi.org/10.1007/s00249-020-01424-1DOI Listing
March 2020

Unusual Two-Step Assembly of a Minimalistic Dipeptide-Based Functional Hypergelator.

Adv Mater 2020 Mar 27;32(9):e1906043. Epub 2020 Jan 27.

School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

Self-assembled peptide hydrogels represent the realization of peptide nanotechnology into biomedical products. There is a continuous quest to identify the simplest building blocks and optimize their critical gelation concentration (CGC). Herein, a minimalistic, de novo dipeptide, Fmoc-Lys(Fmoc)-Asp, as an hydrogelator with the lowest CGC ever reported, almost fourfold lower as compared to that of a large hexadecapeptide previously described, is reported. The dipeptide self-assembles through an unusual and unprecedented two-step process as elucidated by solid-state NMR and molecular dynamics simulation. The hydrogel is cytocompatible and supports 2D/3D cell growth. Conductive composite gels composed of Fmoc-Lys(Fmoc)-Asp and a conductive polymer exhibit excellent DNA binding. Fmoc-Lys(Fmoc)-Asp exhibits the lowest CGC and highest mechanical properties when compared to a library of dipeptide analogues, thus validating the uniqueness of the molecular design which confers useful properties for various potential applications.
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http://dx.doi.org/10.1002/adma.201906043DOI Listing
March 2020

Magnetic Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy.

Langmuir 2020 02 30;36(5):1258-1265. Epub 2020 Jan 30.

Biophysics Program and Department of Chemistry, Macromolecular Science and Engineering, Biomedical Engineering , University of Michigan , Ann Arbor , Michigan 48109-1055 , United States.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state nuclear magnetic resonance (NMR) spectroscopy and aids in the measurement of residual dipolar couplings via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic alignment properties of styrene maleimide quaternary ammonium (SMA-QA) polymer-based nanodiscs by using P and N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic alignment of large-sized (≥20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid bilayer normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs is further shown to flip the alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.
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http://dx.doi.org/10.1021/acs.langmuir.9b03538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414804PMC
February 2020

Expression, purification, and functional reconstitution of F-labeled cytochrome b5 in peptide nanodiscs for NMR studies.

Biochim Biophys Acta Biomembr 2020 05 15;1862(5):183194. Epub 2020 Jan 15.

Biophysics Program, Department of Chemistry, Biomedical Engineering, Macromolecular Science and Engineering, The University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of F NMR experiments to study F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution F NMR experiments. F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular F-F distance constraints to determine the structural interactions between the transmembrane domains.
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http://dx.doi.org/10.1016/j.bbamem.2020.183194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050362PMC
May 2020

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-β Generates Toxic Hetero-oligomers.

J Mol Biol 2020 02 19;432(4):1020-1034. Epub 2019 Dec 19.

Biophysics and Department of Chemistry, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, 48109-1055, USA. Electronic address:

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and β-amyloid (Aβ) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aβ(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aβ(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aβ(M1-42) heteromers comprised of unstructured Aβ(M1-42) and helical 4F. A uniform two-fold reduction in N/H NMR signal intensities of Aβ(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aβ(M1-42) is electrostatically driven and induces unfolding of Aβ(M1-42). Neurotoxicity profiling of Aβ(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aβ(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.
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http://dx.doi.org/10.1016/j.jmb.2019.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054163PMC
February 2020

Exploiting heterogeneous time scale of dynamics to enhance 2D HETCOR solid-state NMR sensitivity.

J Magn Reson 2019 12 14;309:106615. Epub 2019 Oct 14.

Biophysics and Department of Chemistry, Biomedical Engineering, Maromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109-1055, USA. Electronic address:

Multidimensional solid-state NMR spectroscopy plays a significant role in offering atomic-level insights into molecular systems. In particular, heteronuclear chemical shift correlation (HETCOR) experiments could provide local chemical and structural information in terms of spatial heteronuclear proximity and through-bond connectivity. In solid state, the transfer of magnetization between heteronuclei, a key step in HETCOR experiments, is usually achieved using cross-polarization (CP) or insensitive nuclei enhanced by polarization transfer (INEPT) depending on the sample characteristics and magic-angle-spinning (MAS) frequency. But, for a multiphase system constituting molecular components that differ in their time scales of mobilities, CP efficiency is pretty low for mobile components because of the averaging of heteronuclear dipolar couplings whereas INEPT is inefficient for immobile components due to the short T and can yield through-space connectivity due to strong proton spin diffusion for immobile components especially under moderate spinning speeds. Herein, in this study we present two 2D pulse sequences that enable the sequential acquisition of C/H HETCOR NMR spectra for the rigid and mobile components by taking full advantage of the abundant proton magnetization in a single experiment with barely increasing the overall experimental time. In particular, the C-detected HETCOR experiment could be applied under slow MAS conditions, where a multiple-pulse sequence is typically employed to enhance H spectral resolution in the indirect dimension. In contrast, the H-detected HETCOR experiment should be applied under ultrafast MAS, where CP and heteronuclear nuclear Overhauser effect (NOE) polarization transfer are combined to enhance C signal intensities for mobile components. These pulse sequences are experimentally demonstrated on two model systems to obtain 2D C/H chemical shift correlation spectra of rigid and mobile components independently and separately. These pulse sequences can be used for dynamics based spectral editing and resonance assignments. Therefore, we believe the proposed 2D HETCOR NMR pulse sequences will be beneficial for the structural studies of heterogeneous systems containing molecular components that differ in their time scale of motions for understanding the interplay of structures and properties.
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http://dx.doi.org/10.1016/j.jmr.2019.106615DOI Listing
December 2019

Probing protein-protein and protein-substrate interactions in the dynamic membrane-associated ternary complex of cytochromes P450, b, and reductase.

Chem Commun (Camb) 2019 Nov 22;55(89):13422-13425. Epub 2019 Oct 22.

Biophysics and Department of Chemistry, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109-1055, USA.

Cytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b, to metabolize substrates. Using NMR, we reveal changes in the dynamic interplay when all three proteins are incorporated into lipid nanodiscs in the absence and presence of substrates.
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http://dx.doi.org/10.1039/c9cc05904kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879317PMC
November 2019

Metal-Chelated Polymer Nanodiscs for NMR Studies.

Angew Chem Int Ed Engl 2019 11 17;58(48):17246-17250. Epub 2019 Oct 17.

Biophysics Program and Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-1055, USA.

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T ) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene-co-maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu -chelated nanodiscs are demonstrated to reduce the T of protons for both polymer and lipid-nanodisc components. The chelated nanodiscs also decrease the proton T values for a water-soluble DNA G-quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed-up data acquisition from lipid bilayer samples and also to provide structural information from water-soluble biomolecules.
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http://dx.doi.org/10.1002/anie.201910118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861636PMC
November 2019

Use of paramagnetic systems to speed-up NMR data acquisition and for structural and dynamic studies.

Solid State Nucl Magn Reson 2019 10 12;102:36-46. Epub 2019 Jul 12.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; Biophysics, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address:

NMR spectroscopy is a powerful experimental technique to study biological systems at the atomic resolution. However, its intrinsic low sensitivity results in long acquisition times that in extreme cases lasts for days (or even weeks) often exceeding the lifetime of the sample under investigation. Different paramagnetic agents have been used in an effort to decrease the spin-lattice (T) relaxation times of the studied nuclei, which are the main cause for long acquisition times necessary for signal averaging to enhance the signal-to-noise ratio of NMR spectra. Consequently, most of the experimental time is "wasted" in waiting for the magnetization to recover between successive scans. In this review, we discuss how to set up an optimal paramagnetic relaxation enhancement (PRE) system to effectively reduce the T relaxation times avoiding significant broadening of NMR signals. Additionally, we describe how PRE-agents can be used to provide structural and dynamic information and can even be used to follow the intermediates of chemical reactions and to speed-up data acquisition. We also describe the unique challenges and benefits associated with the application of PRE to solid-state NMR spectroscopy, explaining how the use of PREs is more complex for membrane mimetic systems as PREs can also be exploited to change the alignment of oriented membrane systems. Functionalization of membrane mimetics, such as bicelles, can provide a controlled region of paramagnetic effect that has the potential, together with the desired alignment, to provide crucial biologically relevant structural information. And finally, we discuss how paramagnetic metals can be utilized to further increase the dynamic nuclear polarization (DNP) effects and how to preserve the enhancements when dissolution DNP is implemented.
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http://dx.doi.org/10.1016/j.ssnmr.2019.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698407PMC
October 2019

Magnetic Alignment of Polymer Macro-Nanodiscs Enables Residual-Dipolar-Coupling-Based High-Resolution Structural Studies by NMR Spectroscopy.

Angew Chem Int Ed Engl 2019 10 11;58(42):14925-14928. Epub 2019 Sep 11.

Department of Chemistry and Biophysics, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, 48109-1055, USA.

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic-resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic-alignment behavior of lyotropic liquid-crystalline polymer macro-nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high-resolution NMR. The easy preparation of macro-nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.
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http://dx.doi.org/10.1002/anie.201907655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161179PMC
October 2019

Diverse Structural Conversion and Aggregation Pathways of Alzheimer's Amyloid-β (1-40).

ACS Nano 2019 08 24;13(8):8766-8783. Epub 2019 Jul 24.

Institute for Protein Research , Osaka University , Yamadaoka 3-2 , Suita , Osaka 565-0871 , Japan.

Complex amyloid aggregation of amyloid-β (1-40) (Aβ) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aβ aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aβ induced by low solvent polarity accelerated cytotoxic Aβ amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of β-structure-rich protofibrils or oligomers aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aβ aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.
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http://dx.doi.org/10.1021/acsnano.9b01578DOI Listing
August 2019