Protein & Peptide Letters

ISSN: 0929-8665

Protein & Peptide Letters
Volume 13, Number 3, 2006

Contents

Methods to Study Protein Aggregation and Amyloid Formation
Guest Editors: Mireille Dumoulin & Reto Bader


Editorial Pp. 211-212


A Short Historical Survey of Developments in Amyloid Research Pp. 213-217
M. Dumoulin and R. Bader
[Abstract]


Protein Aggregation and Its Consequences for Human Disease Pp. 219-227
C.M. Dobson
[Abstract]


Solid-State NMR as a Probe of Amyloid Structure Pp. 229-234
R. Tycko
[Abstract]


Studying the Natively Unfolded Neuronal Tau Protein by Solution NMR Spectroscopy Pp. 235-246
G. Lippens, A. Sillen, C. Smet, J.-M. Wieruszeski, A. Leroy, L. Buée and I. Landrieu
[Abstract]


Quasielastic Light Scattering Study of Amyloid β-Protein Fibril Formation Pp. 247-254
A. Lomakin and D.B. Teplow
[Abstract]


Insights into Amyloid Fibril Formation from Mass Spectrometry Pp. 255-260
G.L. Caddy and C.V. Robinson
[Abstract]


Amyloid Under the Atomic Force Microscope Pp. 261-270
W.S. Gosal, S.L. Myers, S.E. Radford and N.H. Thomson
[Abstract]


High Pressure Modulates Amyloid Formation Pp. 271-277
J. Torrent, C. Balny and R. Lange
[Abstract]


Combinatorial Approaches to Probe the Sequence Determinants of Protein Aggregation and Amyloidogenicity Pp. 279-286
C. Wurth, W. Kim and M.H. Hecht
[Abstract]


Theoretical Approaches to Protein Aggregation Pp. 287-293
J. Gsponer and M. Vendruscolo
[Abstract]


General Articles


Regular Papers


Structural and Functional Characterization of a Mutant of Pseudocerastes persicus Natriuretic Peptide Pp. 295-300
M.M. Elmi, M. Amininasab, T. Hondo, J. Kikuchi, Y. Kuroda, H. Naderi-Manesh and M.N. Sarbolouki
[Abstract]


Dimerization and Ion Binding Properties of S100P Protein Pp. 301-306
Y. Tutar
[Abstract]


Target Peptide Recognition by S100P Protein and Role of Central Linker Region and Dimer Interface Pp. 307-311
Y. Tutar
[Abstract]


Structural Basis for the Inactivation of AdoMetDC K12R Mutant Pp. 313-317
A. Yerlikaya and B.A. Stanley
[Abstract]


Crystallization Report


Crystallization and Preliminary X-Ray Analysis of the Highly Thermostable Sweet Protein Mabinlin II Pp. 319-321
D.-F. Li, D.-Y. Zhu, Z. Hu and D.-C. Wang
[Abstract]




Abstracts

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Editorial

Protein aggregation has long been experienced as an important problem in the biotechnology industry. It was more recently suggested that a range of disorders including amyloid diseases such as Alzheimer’s and Parkinson’s diseases and type II diabetes, as well as some forms of cancer are associated with protein misfolding and aggregation. Today, it is generally accepted that aberrant protein aggregation results from the failure of proteins to fold or to remain folded in their native state. The fact that protein aggregation plays a prominent role in diseases that are of increasing importance in the context of present-day human health and welfare has stimulated many investigators to focus their work on this process. Defining the kinetic and thermodynamic properties of the aggregation process and characterising at an atomic level the structures of the various species involved in the formation of amyloid fibrils may indeed suggest strategies to prevent or alleviate amyloidoses. These tasks, however, are technically extremely challenging for several reasons. First, the aggregation process is generally irreversible and thereby studies of its kinetic and thermodynamic behaviour are greatly complicated. Second, a suspension of particles scatters the incident light, which generally imposes serious limitations to the use of optical spectroscopy in structural studies of protein aggregates, although fluctuations in the intensity of light scattering over time may under some conditions provide important information on particle size and shape. Finally, the process of protein aggregation and amyloid formation is thought to follow a hierarchical path of assembly involving multiple steps of association and a variety of conformational rearrangements. The heterogeneity and the transient or insoluble nature of the various species seriously limit the applicability of the two most powerful methods of structural biology, namely solution NMR spectroscopy and X-ray diffraction.

As it will become evident from the series of review articles included in this special issue of Protein and Peptide Letters, technical innovations in molecular biology and biophysics have led to a recent blossoming of research devoted to aggregation and amyloid fibril formation, despite all the challenges outlined above. It would clearly be beyond the scope of an issue of this size to give a comprehensive coverage of all techniques that are currently used in this growing field of research. We therefore chose to concentrate on a set of techniques that, in combination with each other, can provide a detailed picture of both kinetic and structural events in protein aggregation and amyloid fibril formation. Each article focuses on a particular technique starting with a general introduction on methodological principles, followed by selected examples that illustrate how it is applied to study mechanistic aspects of peptide and protein assembly.

The scope of the first two reviews is a general overview of the field. Dumoulin and Bader summarize some key discoveries in amyloid research ever since Virchow coined the term “amyloid”, underlying the technical developments that made them possible. Dobson provides a more general overview into protein folding and misfolding and its link to human disease. He reviews our present knowledge of the nature of these fibrillar aggregates and the manner in which they form, and discusses their origins and potential means of suppressing of the pathogenic properties with which amyloid fibrils and their precursors are associated.

Information on the molecular structure of proteins within amyloid fibrils and on the conformational properties of their precursor states will undoubtly lead to a better understanding of the intermolecular interactions by which they are stabilized and the manner in which they form. The first insights into the structure of amyloid fibrils emerged from fibre X-ray diffraction studies which have revealed that all amyloid fibrils are structures rich in β-sheet sharing the so-called cross-β motif as a common core. Two reviews deal with developments in NMR spectroscopy that have enabled structural information of materials in their aggregated and solid states to be obtained. Tycko reviews the recent advances of solid state NMR spectroscopy that have led to high-resolution structural models of amyloid fibrils and in particular those formed by the amyloid-beta (Aβ) peptide. This technique, first introduced by Lansbury and coworkers in the amyloid field, proved particularly valuable as a direct structural probe of amyloid fibrils because it provides constraints on inter-atomic distances and torsion angles at specific sites in non-crystalline materials. Whilst solid state NMR measurements are useful in structural studies of fibrils, solution NMR can provide important information on conformational properties of the monomeric precursor state of an amyloidogenic protein. A number of peptides and proteins involved in amyloid diseases, including the Aβ-peptide, α-synuclein and the Tau protein, are at least partly disordered in their native states. A yet unresolved question concerning these proteins is as to whether and to what extent they exhibit residual structure in specific regions along the polypeptide chain, a problem which can readily be tackled by solution NMR techniques. Lippens et al. present methods to obtain the chemical shift assignment of the natively unfolded Tau441 protein from an appropriate set of multidimensional NMR spectra of and the full-length protein and some protein fragments. They further discuss in some detail how this information can be used to detect residual structure in the Tau protein. Moreover, preliminary NMR data on Tau paired helical filaments (PHFs) suggest experimental ways of mapping the parts of the protein sequence that are involved in the rigid core of the fibrils as compared to those that remain flexible even when the Tau protein is integrated in mature PHFs.

In order to better understand the pathogenic mechanism associated with protein aggregation, it is also essential to obtain insight into the kinetics of the aggregation process. In a comprehensive review Lomakin and Teplow demonstrate how quasielastic light scattering (QLS) can be used to monitor protein aggregation with high sensitivity and resolution. In combination with appropriate methods to analyse the recorded data, fundamental parameters of the protein self-assembly process can be determined, including rate constant values for fibril nucleation and elongation, estimates of both the average fibril length and the activation energy of monomer association.

In the next review, Caddy and Robinson introduce mass spectrometry as a powerful approach to investigate both structural and kinetic aspects of amyloid formation. In particular, the combination of mass spectrometry with hydrogen/deuteriumexchange techniques was successfully used to probe the nature and extent of structural rearrangements that a protein undergoes upon its conversion into amyloid fibrils. In addition, the technique is especially well adapted to detect non-covalently bonded oligomeric species, thus allowing to monitor in real time the aggregation process in its early stages.

The light microscope has been one of the most powerful tools of biologists for centuries and its invention can certainly be said to have revolutionized biology. The first electron microscope was built in 1931 and enabled the cellular components to be visualized at sub-nanometer resolution. Hence, the architecture and dimensions of amyloid fibrils were first determined by electron microscopy (EM). Later, the use of cryo-EM and image processing allowed the reconstruction at 25 Å resolution of a three-dimensional model of an amyloid fibril. The atomic force microscope (AFM), invented in 1986, has some further advantages over the EM. Gosal et al. review the use of this technique applied to protein and peptide self-assembly systems involved in amyloid formation. Unlike the electron microscope, the AFM provides a true three-dimensional surface profile rather than a two-dimensional image of a sample, and it also allows one to measure inter and intra-molecular forces. Moreover, samples viewed by an AFM do not require any special treatment and the AFM can work perfectly well in both air and liquid environment.

Fibril formation in vivo usually takes place over a period of several years. In order to carry out detailed studies of such processes in vitro it is therefore necessary to increase considerably the rates at which they occur. For globular proteins, one way of achieving this objective is to employ conditions that favour the formation of at least partially unfolded states, for example by incubation at low or high pH values, high temperatures, low to moderate concentrations of strong denaturants, in the presence of organic solvents or, more recently, by the use of high hydrostatic pressure. This last technique, which is reviewed by Torrent et al. is known for its capacity to perturb the structure of a protein in a rather gentle manner and consequently for its ability to populate and stabilize partially folded intermediates. Hence, such species can be characterized more easily under high pressure. Depending on the experimental conditions, pressure can also be used to trigger protein aggregation or, on the contrary, to dissociate aggregates. This novel tool has therefore not only proved valuable for in vitro studies of protein misfolding and aggregation, but is currently also drawing increased attention from the pharmaceutical and biotechnology industries to improve the stability of aggregation-prone macromolecules during the production process.

A number of studies suggested that the ability to form amyloid structures is not an unusual feature restricted to a small number of proteins associated with human diseases but is instead a generic property of most - and perhaps all - polypeptide chains. It is clear, however, that the sequence of a peptide or a protein affects its propensity to form amyloid structures under given conditions and that some sequences are far more amyloidogenic than others. This observation raises the question of the nature of the molecular determinants that are responsible for the tendency of these amyloidogenic sequences to assemble into fibrils. In order to investigate the role of a given amino acid on the physico-chemical properties of a protein, one typically mutates it to another naturally occuring amino acid. Wurth et al. carefully review recent advances in applying combinatorial approaches and genetic screens to investigate the sequence determinants of protein aggregation and amyloid formation.

In the last review, Gsponer and Vendruscolo provide an overview on theoretical approaches used to study protein aggregation. These approaches, in combination with experimental observations, are about to produce a unified framework to understand the principles that determine the process of protein aggregation and thus to develop rational strategies to combat it.

In summary, the reviews included in this special issue of Protein and Peptide Letters show that much effort has recently been made to adapt standard techniques in order to follow more subtle and complex aspects of protein aggregation. The special issue also points towards a growing number of links between experiments and theory in various attempts to unify our present picture of protein folding, misfolding and aggregation.

Amyloid fibril formation represents an abberant type of a non-covalent protein-protein interaction and hence, most techniques presented in this issue can be used to study much more general aspects in the regulation of cell function such as signal transduction and transcriptional regulation. Many enzymes, carrier proteins, scaffold proteins and transcription factors function as homo-oligomers thereby giving rise to regulatory properties and cooperative effects in ligand binding. In investigations of such phenomena as well as in studies of protein aggregation, there is usually no single technique that can produce all the necessary information to draw a full picture of the energetics and conformational changes associated with the underlying interactions. Biophysicists will certainly benefit from being familiar with as many of the available techniques as possible in order to obtain detailed information on the molecular mechanisms of amyloid diseases.

Dr. Mireille Dumoulin
Guest Editor
Protein & Peptide Letters
Department of Chemistry
University of Cambridge
Lensfield Road, Cambridge CB2 1EW
UK
E-mail: mmjd3@cam.ac.uk or

Dr. Reto Bader
Guest Editor
Protein & Peptide Letters
Department of Physics
Stockholm University
AlbaNova University Center, 10691 Stockholm,
Sweden
E-mail: bader@physto.se


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A Short Historical Survey of Developments in Amyloid Research
M. Dumoulin and R. Bader

One of the hallmarks of modern science is technically controlled experimentation. In this paper, we underline how technical developments over the last 150 years have repeatedly created new horizons in amyloid research. The main focus is on chemical and biophysical analyses of amyloid fibrils in vivo and in vitro. Investigations into mechanistic aspects of fibril formation and possible links with pathogenesis are also discussed.


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Protein Aggregation and Its Consequences for Human Disease
C.M. Dobson

Protein molecules have emerged through evolution so that they are able to remain in their functional and soluble states under normal physiological conditions, although in other situations they often have a high propensity to aggregate. Aggregation in vivo is associated with a wide range of human disorders, including Alzheimer’s disease and type II diabetes, medical conditions that are becoming increasingly common in the modern world. In such diseases, aggregated proteins can often be observed as highly intractable thread-like species known as amyloid fibrils. This article provides an overview of our present knowledge of the nature of these fibrillar aggregates and the manner in which they form, and discusses the origins and potential means of suppression of the pathogenic properties with which they and their precursors are associated.


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Solid-State NMR as a Probe of Amyloid Structure
R. Tycko

Solid state nuclear magnetic resonance (NMR) has developed into one of the most informative and direct experimental approaches to the characterization of the molecular structures of amyloid fibrils, including those associated with Alzheimer's disease. In this article, essential aspects of solid state NMR methods are described briefly and results obtained to date regarding the supramolecular organization of amyloid fibrils and the conformations of peptides within amyloid fibrils are reviewed.


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Studying the Natively Unfolded Neuronal Tau Protein by Solution NMR Spectroscopy
G. Lippens, A. Sillen, C. Smet, J.-M. Wieruszeski, A. Leroy, L. Buée and I. Landrieu

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer’s diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.


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Quasielastic Light Scattering Study of Amyloid β-Protein Fibril Formation
A. Lomakin and D.B. Teplow

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid β-protein.


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Insights into Amyloid Fibril Formation from Mass Spectrometry
G.L. Caddy and C.V. Robinson

Mass spectrometry has become increasingly important in amyloid research specifically in the mechanism of formation and characterization of fibrils. In this review we highlight key experiments that provide evidence for different conformations, interactions and unfolding intermediates in proteins associated with amyloid diseases.


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Amyloid Under the Atomic Force Microscope
W.S. Gosal, S.L. Myers, S.E. Radford and N.H. Thomson

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.


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High Pressure Modulates Amyloid Formation
J. Torrent, C. Balny and R. Lange

A common mechanism of conformational changes and pathological aggregation of proteins associated with amyloid diseases remains to be proven. High pressure is emerging as a new strategy for studying aspects of amyloid formation. Pressure provides a convenient means to populate and characterize partially folded states, which are thought to have a key role in assembly processes of proteins into amyloid fibrils. High pressure can also be used to dissociate aggregates and amyloid fibrils or on the opposite to generate such species.


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Combinatorial Approaches to Probe the Sequence Determinants of Protein Aggregation and Amyloidogenicity
C. Wurth, W. Kim and M.H. Hecht

Elucidation of the molecular determinants that drive proteins to aggregate is important both to advance our fundamental understanding of protein folding and misfolding, and as a step towards successful intervention in human disease. Combinatorial strategies enable unbiased and model-free approaches to probe sequence/structure relationships. Through the use of combinatorial methods, it is possible (i) to probe the sequence determinants of natural amyloid proteins by screening libraries of amino acid substitutions (mutations) to identify those that prevent amyloid formation; and (ii) to test new hypotheses about the mechanism of formation of amyloid fibrils by using these hypotheses to guide the design of combinatorial libraries of de novo amyloid-like proteins. Here, we review how these two approaches have been used to study the molecular determinants of protein aggregation and amyloidogenicity.


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Theoretical Approaches to Protein Aggregation
J. Gsponer and M. Vendruscolo

The process of protein misfolding and aggregation has been associated with an increasing number of pathological conditions that include Alzheimer’s and Parkinson’s diseases, and type II diabetes. In addition, the discovery that proteins unrelated to any known disorder can be converted into aggregates of morphologies similar to those found in diseased tissue has lead to the recognition that this type of assemblies represents a generic state of polypeptide chains. Therefore, despite the enormous complexity of the in vivo mechanisms that have evolved in living organisms to prevent and control the formation of protein aggregates, the process of aggregation itself appears ultimately to be caused by intrinsic properties of polypeptide chains, in particular by the tendency of the backbone to form hydrogen bonds, and be modulated by the presence of specific patterns of hydrophobic and charged residues. Theoreticians have just recently started to respond to the challenge of identifying the determinants of the aggregation process. In this review, we provide an account of the theoretical results obtained so far.


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Structural and Functional Characterization of a Mutant of Pseudocerastes persicus Natriuretic Peptide
M.M. Elmi, M. Amininasab, T. Hondo, J. Kikuchi, Y. Kuroda, H. Naderi-Manesh and M.N. Sarbolouki

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by ¹H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17-residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.


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Dimerization and Ion Binding Properties of S100P Protein
Y. Tutar

Functional S100P requires dimer formation and dimerization might form for one of the two reasons: i. producing a pair of site for target protein binding or ii. modulation of cation binding affinity. The extent of exposed protein hydrophobicity was related to dimer formation.


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Target Peptide Recognition by S100P Protein and Role of Central Linker Region and Dimer Interface
Y. Tutar

Interaction between S100P and its target protein is an essential step in several cellular functions. The amphiphatic mellitin peptide binds tightly to S100P protein in the presence of calcium cation. Since little is known about the recognition sequence, mellitin interaction form a model for S100P. Interaction between mellitin and protein examined to identify key regions required for the protein-protein interaction.


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Structural Basis for the Inactivation of AdoMetDC K12R Mutant
A. Yerlikaya and B.A. Stanley

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the α (30.7 kDa) and β (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45→ Arg and Lys56→ Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.


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Crystallization and Preliminary X-Ray Analysis of the Highly Thermostable Sweet Protein Mabinlin II
D.-F. Li, D.-Y. Zhu, Z. Hu and D.-C. Wang

Mabinlin II is a thermostable sweet protein isolated from the mature seeds of Capparis masaikai Levl. grown in the subtropical region of the South of China. The Mabinlin II has been crystallized and diffraction data were collected to 1.7 Å resolution. The crystal belongs to space group C2 with unit cell parameters a = 80.11 Å, b = 51.08 Å, c = 47.34 Å, β = 122.77°.