Protein Folding, Misfolding and Aggregation: Classical Themes and Novel Approaches: Volume 13 (RSC Biomolecular Sciences) - Hardcover

Hardcover

ISBN 10: 0854042571 ISBN 13: 9780854042579

Publisher: Royal Society of Chemistry, 2008

Synopsis

This unique book covers all the modern approaches and the many advances experienced in the field of protein folding and aggregation during the last 10 years.

About this title

About the Author

Victor Mu±oz is Associate Professor in Chemistry and Biochemistry at the University of Maryland, USA and Research Professor in Biophysics at the Spanish Research Council (CSIC), Madrid, Spain. He has worked in the area of protein folding and aggregation for the last 16 years and for the last 7 years he has been involved in promoting a change from the classical biochemical to a physical paradigm in experimental protein folding.

From the Back Cover

Protein folding is the process by which newly synthesized proteins fold into the specific three-dimensional structures defining their biologically active states while avoiding aggregation into pathogenic assemblies. It has always been a major focus of research in biochemistry and has often been seen as the unsolved second part of the genetic code. In the last 10 years we have witnessed a quantum leap in the research in this exciting area. Computational methods have improved to the extent of making it possible to simulate the complete folding process of small proteins and the early stages of protein aggregation. Experimental methods have evolved to permit resolution of fast processes of folding reactions and visualisation of single molecules during folding. As a result, the study of protein folding and aggregation has acquired a high level of technical and conceptual sophistication, in which theory, computer simulations and experiments are starting to be combined in a global approach. Protein Folding, Misfolding and Aggregation: Classical Themes and Novel Approaches includes chapters in the areas that have witnessed major developments and are written by top experts in the field. The book is unique in its scope and in its coverage of all the new developments in this area by filling a much needed gap in the current literature. Readers will obtain a unique perspective of the problems faced in the biophysical study of protein conformational behaviour in aqueous solution and how these problems are being solved with a multidisciplinary approach that combines theory, experiment, and computer simulations. RSC The book is essential reading for graduate and postdoctoral students actively involved in protein folding research, other scientists interested in the recent progress of the field, and instructors revamping the protein folding section of their biochemistry and biophysics courses.

Excerpt. © Reprinted by permission. All rights reserved.

Protein Folding, Misfolding and Aggregation

Classical Themes and Novel Approaches

By Victor Muñoz

The Royal Society of Chemistry

Copyright © 2008 Royal Society of Chemistry
All rights reserved.

ISBN: 978-0-85404-257-9

Contents

Preface, v, 
Chapter 1 The α-Helix as the Simplest Protein Model: Helix–Coil Theory, Stability, and Design Andrew James Doig, 
Chapter 2 Kinetics and Mechanisms of α-Helix Formation Urmi Doshi, 
Chapter 3 The Protein Folding Energy Landscape: A Primer Peter G. Wolynes, 
Chapter 4 Hydrogen Exchange Experiments: Detection and Characterization of Protein Folding Intermediates Yawen Bai, 
Chapter 5 Statistical Differential Scanning Calorimetry: Probing Protein Folding–Unfolding Ensembles Beatriz Ibarra-Molero and Jose Manuel Sanchez-Ruiz, 
Chapter 6 Fast Protein Folding Martin Gruebele, 
Chapter 7 Single Molecule Spectroscopy in Protein Folding: From Ensembles to Single Molecules Benjamin Schuler, 
Chapter 8 Computer Simulations of Protein Folding Vijay S. Pande, Eric J. Sorin, Christopher D. Snow and Young Min Rhee, 
Chapter 9 Protein Design: Tailoring Sequence, Structure, and Folding Properties Andreas Lehmann, Christopher J. Lanci, Thomas J. Petty II, Seung-gu Kang and Jeffery G. Saven, 
Chapter 10 Protein Misfolding and β-Amyloid Formation Alexandra Esteras-Chopo, Maria Teresa Pastor and Luis Serrano, 
Chapter 11 Scenarios for Protein Aggregation: Molecular Dynamics Simulations and Bioinformatics Analysis Ruxandra Dima, Bogdan Tarus, G. Reddy, John E. Straub and D. Thirumalai, 
Subject Index, 266, 

CHAPTER 1

The α-Helix as the Simplest Protein Model: Helix — Coil Theory, Stability, and Design

ANDREW JAMES DOIG

Faculty of Life Sciences, The University of Manchester, Jackson's Mill, PO Box 88, Sackville Street, Manchester M60 1QD, UK

1.1 Introduction

Proteins are built of regular local folds of the polypeptide chain called secondary structure. α-Helices are present in nearly all globular proteins, with ≈ 30% of residues found in α-helices. It is such ubiquity and its structural simplicity that makes the α-helix an ideal candidate for detailed quantitative studies of the complex energetic factors involved in protein folding and stability. Here, we discuss structural features of the helix and their contributions to helix stability from studies in peptides. Some earlier reviews in this field are references 2–10.

1.2 Structure of the α-Helix

A helix combines a linear translation with an orthogonal circular rotation. In the α-helix the linear translation is a rise of 5.4 Å per turn of the helix and a circular rotation is 3.6 residues per turn. Side chains spaced i,i + 3, i,i + 4, and i,i + 7 are therefore close in space and interactions between them can affect helix stability. Spacings of i,i + 2, i,i + 5, and i,i + 6 place the side chain pairs on opposite faces of the helix avoiding any interaction. The helix is primarily stabilized by i,i + 4 hydrogen bonds between backbone amide groups.

The conformation of a polypeptide can be described by the backbone dihedral angles Φ and ψ. Most Φ, ψ combinations are sterically excluded, leaving only the broad β region and narrower α region. The residues at the N-terminus of the α-helix are called N'-N-cap-N1-N2-N3-N4 etc., where the N-cap is the residue with non-helical Φ, ψ angles immediately preceding the N-terminus of an α-helix and N1 is the first residue with helical Φ, ψ angles. The C-terminal residues are similarly called C4-C3-C2-C1-C-cap-C etc. The N1, N2, N3, C1, C2, and C3 residues are unique because their amide groups participate in i,i + 4 backbone–backbone hydrogen bonds using either only their CO (at the N-terminus) or NH (at the C-terminus) groups. The need for these groups to form hydrogen bonds has powerful effects on helix structure and stability.

1.2.1 Capping Motifs

The amide NH groups at the helix N-terminus are satisfied predominantly by side-chain H-bond acceptors. In contrast, carbonyl CO groups at the C-terminus are satisfied primarily by backbone NH groups from the sequence following the helix. The presence of such interactions would therefore stabilize helices. These interactions can be identified as specific patterns found at or near the ends of helices and are generally termed capping motifs.

A common pattern of capping at the helix N-terminus is the capping box. Here, the side chain of the N-cap forms a hydrogen bond with the backbone of N3 and, reciprocally, the side chain of N3 forms a hydrogen bond with the backbone of the N-cap. The definition of the capping box was expanded by Seale et al. to include an associated hydrophobic interaction between residues N' and N4 and is also known as a 'hydrophobic staple'. A variant of the capping box motif is termed the "big" box with an observed hydrophobic interaction between non-polar side-chain groups in residues N4 and N" (not N'). The Pro-box motif involves three hydrophobic residues and a Pro residue at the N-cap.

The two primary capping motifs found at helix C-termini are the Schellman and the al motifs. The Schellman motif is defined by a doubly hydrogen-bonded pattern between backbone partners, consisting of hydrogen bonds between the amide NH at C" and the carbonyl CO at C3 and between the amide NH at C' and the carbonyl CO at C2, respectively. The associated hydrophobic interaction is between C3 and C". In a Schellman motif, polar residues are highly favoured at the C1 position and the C' residue is typically glycine. If C" is polar, the alternative αL motif is observed, defined by a hydrogen bond between the amide NH at C' and the carbonyl CO at C3. As in the Schellman motif, the C' residue is typically glycine, which adopts a positive value of Φ. However, the hydrophobic interaction in an αL is heterogeneous, occurring between C3 and any of several residues external to the helix (C3', C4', or C5').

A notable difference between the N- and C-terminal motifs is that at the N-terminus, helix geometry favors side-chain-to-backbone hydrogen bonding and selects for compatible polar residues. Accordingly, the N-terminus promotes selectivity in all polar positions, especially N-cap and N3 in the capping box. In contrast, at the C-terminus, side-chain-to-backbone hydrogen bonding is disfavored. Backbone hydrogen bonds are satisfied instead by post-helical backbone groups. The C-terminus need only select for C' residues that can adopt positive values of the backbone dihedral angle Φ, most notably Gly.

1.2.2 Metal Binding

One way to stabilize helix conformations, especially in short peptides, is to introduce an artificial nucleation site composed of a few residues fixed in a helical conformation. For example, the calcium-binding loop from EF-hand proteins saturated with a lanthanide ion promotes a rigid short helical conformation at its C-terminus region. This system has been used to measure enthalpic terms contributing to helical preferences of the amino acids. In the presence of Cd ions, a synthetic peptide containing Cys-His ligands i,i + 4 apart at the C-terminal region increased helicity (that is the average probability of finding dihedral angle pairs in values typical of α-helix) from 54% to 90%. The helicity of a similar peptide containing His-His ligands increased by up to 90% as a result of Cu and Zn binding. The addition of a cis-Ru(III) ion to a 6-mer peptide, Ac-AHAAAHA-NH2, changed the peptide conformation from random coil to 37% helix. An 11-residue peptide was converted from random coil to 80% helix content by the addition of Cd ions, although the ligands used were not natural amino acids but aminodiacetic acids. As(III) stabilizes helices when bound to Cys side chains spaced i,i + 4 by -0.7 to -1.0 kcal mol-1. 19-Membered metallocyclic rings induce helix formation by covalently linking helical turns.

1.2.3 The 310-Helix

310-Helices are stabilized by i,i +3 hydrogen bonds, instead of the i,i + 4 found in α-helices, making the cylinder of the 310-helix narrower than α and their hydrogen bonds non-linear. 3–4% of residues in crystal structures are in 310-helices. Most 310-helices are short, only 3 or 4 residues long, compared to a mean of 10 residues in α-helices, and are commonly found as N- or C-terminal extensions to an a-helix: strong amino acid preferences have been observed for different locations within the interior and N- and C-caps of 310-helices in crystal structures. The 310-helix is being recognized as of increasing importance in isolated peptides and even as a possible intermediate in α-helix formation.

1.2.4 The π-Helix

In contrast to the widely occurring α- and 310-helices, the π-helix is extremely rare. The π-helix is unfavorable for three reasons: its dihedral angles are energetically unfavorable relative to the α-helix, its three-dimensional structure has a 1 Å hole down the center that is too narrow for access by a water molecule resulting in the loss of van der Waals interactions, and a higher number of residues (four) must be correctly oriented before the first i,i + 5 hydrogen bond is formed, making helix initiation more entropically unfavorable than for α- or 310-helices. π-Helices are known in both peptide and proteins, however.

1.3 Design of Peptide Helices

The earliest work on peptide helices was on long homopolymers of Glu or Lys which show coil-to-helix transitions on changing the pH from charged to neutral. The neutral polypeptides are metastable and prone to aggregation, ultimately to β-sheet amyloid. In 1971 Brown and Klee reported that the C-peptide of ribonuclease A, which contains the first 13 residues of the protein and which forms a helix in the protein, had high helical content at 0°C. Work on the C-peptide showed that the replacement of interior helical residues with Ala was stabilizing, indicating that a major reason why this helix was folded in isolation was the presence of three successive alanines from positions 4–6. This led to the successful design of isolated, monomeric helical peptides in aqueous solution, first containing several salt bridges and a high alanine content, based on (EAAAK)n and then a simple sequence with a high alanine content solubilized by several lysines. These 'AK peptides' are based on the sequence (AAKAA)n, where n is typically 2–5. The Lys side chains are spaced i,i + 5 so they are on opposite faces of the helix, giving no charge repulsion. Hundreds of AK peptides have been studied, giving most of the available results on helix stability in peptides. The alanines in the (EAAAK)n-type peptides may be removed entirely; E4K4 peptides, with sequences based on (EEEEKKKK)n or EAK patterns, are also helical, stabilized by large numbers of salt bridges.'

1.3.1 Host–Guest Studies

Extensive work from the Scheraga group has obtained helix–coil parameters using a host–guest method. Long random co-polymers were synthesized of a water soluble, non-ionic guest (poly[N5-(3 -hydroxypropyl)-L-glutamine] (PHPG) or poly[N5 -(4-hydroxybutyl)-L-glutamine] (PHBG)), together with a low (10–50%) content of the guest residue. Using the s and σ Zimm–Bragg helix–coil parameters (see below) for the host homopolymer, it was possible to calculate those for the guest using helix–coil theory as a function of temperature. The results obtained from the host–guest work are in disagreement with most of the results from short peptides of fixed sequence.

1.3.2 Helix Lengths

Helix formation in peptides is cooperative, with a nucleation penalty. Helix stability therefore tends to increase with length, in homopolymers at least. As the length of a homopolymer increases, the mean fraction helix will level off below 100%, as long helices tend to break in two. In heteropolymers, observed lengths are highly sequence dependent. As helices are at best marginally stable in monomeric peptides in aqueous solution, they are readily terminated by the introduction of a strong capping residue or a residue with a low intrinsic helical preference.

The length distribution of helices in proteins is very different from homo- and heteropolymers. Most protein helices are short, with 5 to 14 residues most abundant. There is a general trend for a decrease in frequency as the length increases beyond 13 residues. Helix lengths longer than 25 are rare. There is also a preference to have close to an integral number of turns so that their N- and C-caps are on the same side of the helix.

1.3.3 The Helix Dipole

The secondary amide group in a protein backbone is polarized with the oxygen negatively charged and hydrogen positively charged. In a helix, the amides are all oriented in the same direction with the positive hydrogens pointing to the N-terminus and negative oxygens pointing to the C-terminus. This can be regarded as giving a partial positive charge at the helix N-terminus and a partial negative charge at the helix C-terminus. In general, therefore, negatively charged groups are stabilizing at the N-terminus and positively charged at the C-terminus. An alternative interpretation of these results is that favored side chains are those that can make hydrogen bonds to the free amide NH groups at N1, N2, and N3 or free CO groups at C1, C2, and C3. Charged groups can form stronger hydrogen bonds than neutral groups, thus providing an alternative rationalization of the pH titration results. These hypotheses are not mutually exclusive, as a charged side chain can also function as a hydrogen bond acceptor or donor. Measurements of the amino acid preferences for the N-cap, N1, N2, and N3 positions in the helix allow a comparison to be made of the relative importance of helix dipole and hydrogen bonding interactions, suggesting that both charge and hydrogen-bonding interactions are important.

1.3.4 Acetylation and Amidation

A simple, yet effective, way to increase the helicity of a peptide is to acetylate its N-terminus. Acetylation removes the positive charge that is present at the helix terminus at low or neutral pH; this charge would interact unfavorably with the positive helix dipole and free N-terminal NH groups. The extra CO group from the acetyl group can form an additional hydrogen bond to the NH group, putting the acetyl at the N-cap position. This has a strong stabilizing effect by approximately 1.0 kcal mol-1 compared to alanine.

Amidation of the peptide C-terminus is structurally analogous to N-terminal acetylation: the helix is extended by one hydrogen bond and an unfavorable charge–charge repulsion with the helix dipole is removed. The energetic benefit of amidation is rather smaller, however, with the amide group being no better than Ala and in the middle if the C-cap residues are ranked in order of stabilization effect. As most helical peptides studied to date are both acetylated and amidated, and acetylation is more stabilizing than amidation, the distribution of helicity along the peptide is generally skewed so that residues near the N-terminus are more helical than those near the C-terminus.

1.3.5 Solubility

Peptide aggregation can be assayed rigorously by sedimentation equilibrium, which determines the oligomeric state of a molecule in solution. This is difficult, however, with the short peptides often used as their molecular weights are at the lower limit for this technique. A simpler method is to check a spectroscopic signal that depends on peptide structure, most obviously circular dichroism (CD), as a function of concentration. If the signal depends linearly on peptide concentration across a large range, including that used to study the peptide structure, it is safe to assume that the peptide is monomeric. An oligomer that does not change state, such as a coiled-coil, across the concentration range cannot be excluded, however. Light scattering can detect aggregation. A monomeric peptide should have a flat baseline in a UV spectrum outside the range of any chromophores in the peptide. Stock solutions of a peptide with a single tyrosine isolated from the helix region by Gly should have A300/ A275<0.02 and A250< A275<0.2.

Consideration of solubility is essential when designing helical peptides. Solubility can be achieved most easily by including polar side chains spaced i,i + 5 in the sequence where they cannot interact. Lys, Arg and Gln are used most often for this purpose. Gln may be preferred if unwanted interactions with charged Lys or Arg may be a problem, but some AQ peptides lack sufficient solubility and AQ peptides are less helical.

The spacing of side chains in the helix is best visualized with a helical wheel, to ensure that the designed helix does not have a non-polar face that may lead to dimerization. The following webpage provides a useful resource for this: http://cit.itc.virginia.edu/~cmg/Demo/wheel/wheelApp .html

1.3.6 Concentration Determination

An accurate measurement of helix content depends on an accurate spectroscopic measurement and, equally importantly, peptide concentration. This is usually achieved by including a Tyr side chain at one end of the peptide. The extinction coefficient of Tyr at 275 nm is 1450 M-1 cm-1. If Trp is present, measurements at 281 nm can be used where the extinction coefficient of Trp is 5690 M-1 cm-1 and Tyr 1250 M-1 cm-1. Though the inclusion of aromatic residues is required for concentration determination, this can have the unwanted side effect of perturbing a CD spectrum, leading to an inaccurate determination of helix content. A simple solution to this problem is to separate the terminal Tyr from the rest of the sequence by one or more Gly residues. If the aromatic residues must be included within the helical region, the CD spectrum should be corrected to remove this perturbation.

(Continues...)

Excerpted from Protein Folding, Misfolding and Aggregation by Victor Muñoz. Copyright © 2008 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2008
• Language: English
• ISBN 10: 0854042571
• ISBN 13: 9780854042579
• Binding: Hardcover
• Number of pages: 288
• Editor: Victor Muñoz