d位置胺基酸的側鏈結構對於雙螺旋穩定度的影響

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Department of Chemistry College of Science

National Taiwan University Master Thesis

Effect of Side Chain Structure of d-Position Residue on Coiled Coil Stability

YI-JEN WENG

Advisor: Richard Ping Cheng, Ph.D.

January, 2013

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(5 timers at a time) paper

-- --

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α

d

GCN4 d

(Leu12) IaLd

Es, MR, [L, B1, B5]

d

α

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Abstract

Coiled coil is a superhelical twist formed by two to five wrapping α-helices. It is a

common structural motif that can be found in transcription factors, cytoskeletal systems,

contractile systems and etc. These biochemical roles rely on well-defined and stable

structures of coiled coils. Accordingly, the effect of side chain structure of d-position

residue, which is buried in the coiled coil interface, on coiled coil stability was

investigated. GCN4 coiled coil was employed, of which the 2^nd d-residue (Leu12) was

substituted with various amino acids. IaLd coiled coils were used to obtain the coiled

coil propensities of these amino acids. Guanidinium denaturation of the coiled coils was

monitored by circular dichroism spectroscopy. Free energy of unfolding was derived

from the guanidinium denaturation data. The hydrophobicities of the amino acids were

measured by thin layer chromatography. Structural parameters Es, MR, [L, B1, B5], and

side chain volume were employed in the analysis. Results show that the shape and size

of the residue side chain contribute more than hydrophobicity to the coiled coil stability

in coiled coils with the d-position residue bearing aliphatic side chains.

Keywords: coiled coil, GCN4, helix propensity, amino acid hydrophobicity, side chain structure

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List of Figures

Figure 1-1. Dihedral angles φ and ψ ... 3!

Figure 1-2. Ramachandran plot. ... 3!

Figure 1-3. Four levels of protein structure. ... 4!

Figure 1-4. A hydrogen bond. ... 7!

Figure 1-5. The α-helix backbone hydrogen bond. ... 9!

Figure 1-6. Illustration of a coiled coil. ... 11!

Figure 2-1. GCN4-DNA complex. ... 18!

Figure 2-2. Illustration of knobs-into-hole interactions, parallel packing, and perpendicular packing. ... 22!

Figure 2-3. Structures of leucine, trifluoroleucine, and hexafluoroleucine. ... 25!

Figure 2-4. The sequence and the helical wheel diagram of GCN4.. ... 29!

Figure 2-5. CD spectrum of GCN4-Phe2. ... 29!

Figure 2-6. Chemical structures of the amino acids. ... 30!

Figure 2-7. CD spectra of GCN4-Xaa peptides at 30 µM peptide concentration in 50 mM phosphate and 150 mM NaCl buffer at pH 7 and 4 °C ... 34!

Figure 2-8. A typical guanidinium titration curve with the proper folded and unfolded baselines depicted. ... 38!

Figure 2-9. Guanidinium denaturation curves for GCN4-Xaa peptides at 30 µM peptide in 50 mM phosphate, 150 mM NaCl, and 0 M to 6 M (with 0.1 M intervals) guanidinium chloride at pH 7 and 4 °C as monitored by CD at 222 nm reported in mean residue ellipticity. ... 39!

Figure 2-10. Fraction unfolded as a function of guanidinium concentration for GCN4-Xaa peptides as derived from the guanidinium denaturation curves. ... 40!

Figure 2-11. The plot of ΔGunfold against guanidinium concentration for GCN4-Tba .. 44!

Figure 2-12. The bar graph of ΔGunfold, H2O of GCN4 peptides. ... 44!

Figure 2-13. ΔGunfold of GCN4-Leu and GCN4-Tba. ... 46!

Figure 2-14. Sequence and helical wheel of IaLd. ... 48!

Figure 2-15. CD spectra of IaLd-Xaa at 20 µM peptide in 10 mM MOPS at pH 7.5 and 25 °C. ... 51!

Figure 2-16. Guanidinium denaturation curves for IaLd-Xaa peptides at 20 µM peptide concentration in 10 mM MOPS with 0 to 6 M (at 0.1 M intervals)

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guanidinium chloride at pH 7.5 and 25 °C as monitored by CD at 222 nm reported in mean residue ellipticity ... 55!

Figure 2-17. Fraction unfolded as a function of guanidinium concentration for

IaLd-Xaa peptides as derived from guanidinium denaturation curves. ... 56!

Figure 2-18. The bar graph of ΔGunfold, H2O of IaLd peptides. ... 57!

Figure 2-19. Regressions between the residual energy and log Pow, Es, MR, and side chain volume. ... 62!

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List of Tables

Table 2-1. Crude Yield of Peptide Synthesis, Molecular Formula, Calculated [MH⁺], and Observed m/z of GCN4 Peptides ... 32!

Table 2-2. Concentrations and Regression Coefficients of GCN4 Peptides ... 33!

Table 2-3. [θ]222 at 0 M Guanidinium, Melting Concentrations ([C]m), m values, and ΔGunfold, H2O of GCN4 Peptides ... 35!

Table 2-4. Crude Yield of Peptide Synthesis, Molecular Formula, Calculated [MH⁺], and Observed m/z of IaLd Peptides ... 49!

Table 2-5. Concentrations and Regression Coefficients of IaLd Peptides ... 50!

Table 2-6. [θ]222 at 0 M Guanidinium, Melting Concentrations ([C]m), m values, and ΔGunfold, H2O of IaLd Peptides ... 52!

Table 2-7. log Pow of the Amino Acids ... 58!

Table 2-8. Es, [L, B1, B5], MR, and the Volume of the Amino Acids Side Chains ... 60!

Table 2-9. Equations and R values of the Regressions Between Residual Energy, log Pow Es, MR, [L, B1, B5], and Side Chain Volume ... 62!

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Abbreviations

Abu Aminobutyric aicd

Allo Ile Alloisoleucine

Arg Arginine

Asn Asparagine

Asp Aspartate

CaM Calmodulin

CD Circular dichroism

Cha Cyclohexylalanine

Cpa Cyclopentylalanine

DIEA Diisopropylethylamine

DMF Dimethylformamide

DNA Deoxyribonucleic acid

ESI-MS Electrospray ionization mass spectrometry Fmoc N-9-Fluorenylmethoxycarbonyl

Fmoc-Osu N-(9-Fluorenylmethoxycarbonyloxy) succinimide

Gln Glutamine

Glu Glutamate

Gly Glycine

HBTU O-1H-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluoro phosphate

Hfl Hexafluoroleucine

His Histidine

HOBt 1-Hydroxybenzotriazole

Ile Isoleucine

KIH Knobs-into-holes

Leu Leucine

Lys Lysine

MALDI-TOF MS Matrix-assisted laser desorption ionization time-of-flight

Met Methionine

MOPS 3-(N-morpholino)propanesulfonic acid

MR Molecular refractivity

MRE Mean residue ellipticity

MW Molecular weight

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Nle Norleucine

NMR Nuclear magnetic resonance

Nva Norvaline

Pff Pentafluorophenylalanine

Phe Phenylalanine

QSAR Quantitative structure–activity relationship

RNA Ribonucleic aicd

RP-HPLC Reverse phase-high performance liquid chromatography

Ser Serine

SPPS Solid phase peptide synthesis

Tba tert-Butylalanine

TFA Trifluoroacetic acid

Tfl Trifluoroleucine

THF Tetrahydrofuran

Tyr Tyrosine

TLC Thin layer chromatography

UV-vis Ultraviolet-visible

Val Valine

VBP Vitellogenin binding protein

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Chapter 1 Introduction

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Proteins

Proteins are one of the most versatile and most abundant macromolecules in living

systems.^{1, 2} Proteins serve as the downstream terminus of the central dogma of

molecular biology, which describes the direction of the flow of genetic information.³

The genetic information stored in deoxyribonucleic acid (DNA) is passed down to

ribonucleic acid (RNA) by transcription.⁴ RNA then serves as the template in the

translation process, leading to the synthesis of proteins.⁵ These proteins then participate

in a variety of physiological processes, such as catalysis,⁶ immune response,⁷ signal

transduction,⁸ and construction and support of cellular structures.⁹ Proteins are

important to living systems, and studies to enhance fundamental knowledge on proteins

should facilitate our understanding of how Nature functions, and also lead to

unprecedented technological advancements.

A protein is a biopolymer composed of amino acids.² There are twenty naturally

occurring amino acids, nineteen of which are L-α amino acids bearing different side

chains. These side chains have various functional groups and properties, and play

different roles in protein functions. The remaining amino acid is glycine, which has no

side chain and is therefore achiral. Amino acids are connected by an amide bond

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between the α-amino group of one amino acid and the α-carbonyl group of the other,

yielding a polypeptide chain. Due to resonance, the amide bond has considerable double

bond character, limiting bond rotation and therefore restricting the conformation of the

peptide backbone. Dihedral angles are defined along the peptide backbone. The dihedral

angle between Ccarbonyl - N plane and Cα - Ccarbonyl plane is defined as phi (φ), and the

dihedral angle between N - Cα plane and Ccarbonyl - N plane is defined as psi (ψ) (Figure

1-1).¹⁰ A plot of ψ against φ is called Ramachandran plot (Figure 1-2).¹⁰ The shaded

region in the plot shows the distribution of φ and ψ found in several structures (Figure

1-2).¹⁰

Figure 1-1. Dihedral angles φ and ψ.

Figure 1-2. Ramachandran plot.¹⁰

NH O

R ψ

φ

Figure 1-4. Ramachandran plot for various secondary structures.¹⁹ Protein Secondary Structure

The polypeptide chain can form local regularly repeating structures called secondary structure. These structures are formed by hydrogen bonds between NH and carbonyl groups in the backbone. Common secondary structure motifs include α-helix, β-sheet, β-turn, and Ω-loop.^{18, 20-24} An α-helix is a right-handed rod-like structure.¹⁸ The left-handed helix is very rare due to the unfavorable steric clash between the side chains and the backbone.¹⁸ A β-sheet is a relatively flat with some right-handed twist structure.²² A β-sheet is composed of two or more β strands in either antiparallel or parallel orientation.²² The compact and globular shape of most proteins can be attributed to the presence of reverse turns (aka: β-turn, hairpin bends) which promotes the chain reversal of polypeptide chain.^{24, 25} Reverse turns are stabilized by hydrogen bonds between the carbonyl group of residue i and the NH of residue i+3.

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Protein Structures

The structure of a protein is critical for its biological function. A protein performs its

function only when it is correctly folded.¹¹ Upon losing its structure, for instance, being

denatured by heat or chemical denaturant, a protein becomes inactive.¹² The structure of

a protein is categorized into four hierarchical levels: primary, secondary, tertiary, and

quaternary structures (Figure 1-3). Primary structure refers solely to the amino acid

sequence of the protein, without consideration of the three dimensional structures of the

polypeptide chain.¹³

Figure 1-3. Four levels of protein structure. (Tertiary structure: calmodulin, protein data bank code 1CLL. Quaternary structure: fibrinogen, protein data bank code 3GHG.)

Secondary structures are local structural motifs composed of regularly repeating

spatial arrangement of the residues. Secondary structures can be defined by the pattern

of hydrogen bonds or backbone dihedral angles of the peptide chain.^{14, 15} For example,

α-helix, a common secondary structure, is characterized by the main-chain hydrogen

bond between the carbonyl oxygen and the amide hydrogen located four residues away Primary

LEDKVEE

Secondary

2ZTA

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Tertiary

1CLL

Page 1 of 1 Printed at 00:01:19 on Fri Mar 2 2012

Quaternary

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in the sequence.¹⁶ The dihedral angles (φ, ψ) of an α-helix is defined to be (-57°, -47°)

(Figure 1-2).¹⁰ Another common secondary structure, β-sheet, is made of two or more β

strands that are connected by hydrogen bonds between backbone carbonyl oxygen and

the amide hydrogen.¹⁷ The dihedral angles (φ, ψ) of a β-sheet are (-119°, +113°) for

parallel sheet and (-139°, +135°) for antiparallel sheet.¹⁰

Tertiary structure refers to the overall structure of one polypeptide chain, containing a

combination of several secondary structures. A typical tertiary structure has nonpolar

residues buried in the interior, making the hydrophobic core.¹⁸ Polar and charged

residues are usually found on the surface, where the protein is in contact with the

aqueous surrounding.¹⁸ Calmodulin (CaM) is an example of tertiary structure, which is

a protein that binds calcium ion and evokes downstream biochemical reactions. CaM is

a 148-amino-acid polypeptide chain made up of several α-helices and turns.¹⁹

Quaternary structure is the spatial arrangement of several polypeptide chains. One

example is the fibrinogen, a protein found in blood and is involved in blood coagulation.

Fibrinogen consists of six chains, (αβγ)2, in which α, β, and γ are the differernt

subunits.²⁰

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Forces in Protein Folding

The forces that dictate protein folding include electrostatic interactions, hydrogen bond,

hydrophobic effect, and van der Waal’s interactions.²¹ The first two occur in charged

and polar groups, whereas the last two are important for nonpolar entities. Electrostatics

include charge to charge, charge to dipole, and dipole to dipole interactions. The

charges originate from the following amino acids: aspartic acid (Asp), glutamic acid

(Glu), lysine (Lys), arginine (Arg), and to a much lesser content, histidine (His). At

physiological pH (pH 7.4), side chains of Asp and Glu are deprotonated and are

negatively charged. On the other hand, Lys, Arg, and His are protonated and bear a

positive charge. Opposite charges attract one another, resulting charge-charge

interactions between these amino acids. A dipole is the asymmetric distribution of

election density along a chemical bond caused by the difference in electronegativity of

atoms. Dipoles can be attracted by ions or other dipoles on the protein, leading to

dipole-charge and dipole-dipole interactions. Electrostatic interactions may provide

extra stability to protein structures, as in the case of hyperthermophilic proteins.²² These

proteins are able to withstand high temperatures, and this stability originates from the

numerous electrostatic interactions between the charged residues on the protein

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surface.²²

A hydrogen bond is the consequence of the interaction between a hydrogen bond

donor (HX) and a hydrogen bond receptor (Y) (Figure 1-4). The atoms X and Y could

be nitrogen, oxygen, or fluorine atoms, which are all highly electronegative atoms. The

electronegativity of X causes uneven distribution of electron density along the H-X

bond, resulting in a partial positive charge on H. This charge is attracted to the

nonbonding electrons of Y, therefore forming a hydrogen bond. Hydrogen bond is the

dominant force for the formation of secondary structures. The deprival of these

hydrogen bonds (e.g., using solvents like trifluoroacetic acid or formic acid, which

protonate the peptide backbone) diminishes peptide structure.^{23, 24}

Figure 1-4. A hydrogen bond.

Hydrophobic effect is the release of water molecules around a hydrophobic group

when two or more such groups approach one another.²⁵ Water molecules that are in

contact with nonpolar solutes are, to some extent, orderly arranged.²⁵ When the

nonpolar groups come into contact with one another, these ordered water molecules are

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X H Y

δ

⁻

δ

⁻

δ

⁺

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example of the importance of the hydrophobic effect in protein folding is the coiled coil

motif. A coiled coil is composed of intertwining α-helices.²⁶ The residues in helices

interface are mainly hydrophobic.^{26, 27} Substituting the hydrophobic residues with polar

residues destablizes the coiled coil.²⁷

van der Waal's interaction (or London dispersion force) is the attractive force

between apolar molecules.²¹ The electron density distribution of the atoms in apolar

group may fluctuate, generating an instantaneous dipole. This dipole may induce an

opposite dipole in a nearby molecular entity. The attraction between these dipoles is

termed London dispersion force. This is a weak interaction and is significant only when

the apolar groups are close in distance. The huge number of London dispersion forces in

proteins make them an important driving force in protein folding.

α-Helix

α-Helix is a common secondary structure that constitutes about 30% of all protein

structures known to date.^{15, 28} The naturally occurring α-helix is a right-handed, rod-like

structure characterized by consecutive, main-chain, i i + 4 hydrogen bonds between

each carbonyl oxygen (i) and an amide hydrogen (i+4) on the adjacent helical turn

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(Figure 1-5).¹⁶ A helix has backbone dihedral angles (φ, ψ) around (-57°, -47°) and has

3.6 residues per turn.^{10, 16} Many factors determine the stability of a helix, one of which

is the helix propensity of the constituting amino acids. Chou and Fasman analyzed the

frequency of appearance of each amino acid in helices.¹⁵ The statistics revealed that

different amino acids have different probabilities to be found in a helix. The

thermodynamic tendency to form a helix can be described in a statistical mechanical

manner in Lifson-Roig theory, in which helix propensity (w) of an amino acid is

employed to describe the equilibrium between α-helix and random coil.²⁹ The greater

the helix propensity, the greater the probability to find an amino acid in the helical state.

Helices are often combined with other secondary structures to form motifs, which

usually exhibit certain functions.¹ For example, the helix-turn-helix motif is a DNA

binding motif,³⁰ and the helix-loop-helix ("EF hand") is a calcium ion binding motif.¹⁹

Figure 1-5. The α-helix backbone hydrogen bond.

NH HN

O

NH

O H

N O

O NH

O HN

R_i R_i+2 R_i+4

R_i+1 R_i+3

i to i+4 hydrogen bond

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Coiled coil

Coiled coils are formed by 3-5% of all amino acids in proteins,³¹ and can be found in

transcription factors,³² cytoskeletal³³ and contractile³⁴ systems, viral envelope

proteins,³⁵ and other systems.³⁶ A coiled coil is formed by two to five α-helices

wrapping around each other to form a slight left-handed superhelical twist (Figure

1-6).²⁶ The sequence of a coiled coil is characterized by the “heptad repeat”, abcdefg, in

which positions a and d are predominantly hydrophobic residues, such as leucine (Leu)

and isoleucine (Ile), leading to a hydrophobic interface between two wrapping helices.

In contrast, the e and g positions are frequently charged residues that form interhelical

ion pairs.²⁶ The three dimensional architecture was first proposed by Crick in 1953 as

the “knobs into holes” model, which states that a coiled coil is stabilized by the packing

of hydrophobic side chains “knobs” into the “holes” that are the space between

hydrophobic side chains of the neighboring helix.³⁷ According to the type and

orientation of the monomers, coiled coils can be further categorized into

homodimer/heterodimer and parallel/antiparallel coiled coils.³⁸ A homodimer is made

up of two identical monomers, whereas a heterodimer consists of two different

monomers. A parallel coiled coil has the N-termini of the two helices located on the

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same end, whereas an antiparallel coiled coil has the two N-termini located at opposite

ends.

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Figure 1-6. Illustration of a coiled coil. (a) A coiled coil. (GCN4 leucine zipper. Protein data bank code 2ZTA.) (b) The helical wheel of a coiled coil.

Thesis Overview

Coiled coil is a common structure in proteins. The factors contributing to coiled coil

stability include hydrophobicity of the coiled coil interface, coiled coil propensity of the

constituting amino acids, and the packing (or the van der Waal's interacitons) of the

residues at the interface.³⁹ Studies involving natural amino acids have shown that

geometric properties of the buried amino acids can influence the stability of coiled

coils.^{39, 40} To further elucidate the relationship between structure, hydrophobicity, and

coiled coil stability, natural and non-natural amino acids with various side chain

structures and properties were introduced into different coiled coil systems. Also, the

hydrophobicities of the amino acids were measured. Several structural parameters were

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Reference

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16.! Pauling, L.; Corey, R. B. The structure of synthetic polypeptides. Proc. Natl. Acad.

Sci. U. S. A. 1951, 37, 241-250.

17.! Pauling, L.; Corey, R. B. The pleated sheet, a new layer configuration of

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polypeptide chains. Proc. Natl. Acad. Sci. U. S. A. 1951, 37, 251-256.

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19.! Chattopadhyaya, R.; Meador, W. E.; Means, A. R.; Quiocho, F. A. Calmodulin structure refined at 1.7 A resolution. J. Mol. Biol. 1992, 228, 1177-1192.

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23.! Hanlon, S.; Klotz, I. M. Protonation of polypeptides in "helix-breaking" solvents:

spectral and optical-rotatory properties in solutions containing strong organic acids. Biochemistry 1965, 4, 37-48.

24.! Lotan, N.; Bixon, M.; Berger, A. Alpha-helix formation by solvent-solvent interaction. Biopolymers 1967, 5, 69-77.

25.! Southhall, N. T.; Dill, K. A.; Haymet, A. D. A View of the Hydrophobic Effect. J.

Phys. Chem. B 2002, 106, 521-533.

26.! Mason, J. M.; Arndt, K. M. Coiled coil domains: Stability, specificity, and biological implications. ChemBioChem 2004, 5, 170-176.

27.! Gonzalez, L., Jr.; Woolfson, D. N.; Alber, T. Buried polar residues and structural specificity in the GCN4 leucine zipper. Nat. Struct. Biol. 1996, 3, 1011-1018.

28.! Cheng, R. P.; Girinath, P.; Suzuki, Y.; Kuo, H. T.; Hsu, H. C.; Wang, W. R.; Yang, P. A.; Gullickson, D.; Wu, C. H.; Koyack, M. J.; Chiu, H. P.; Weng, Y. J.; Hart, P.;

Kokona, B.; Fairman, R.; Lin, T. E.; Barrett, O. Positional effects on helical Ala-based peptides. Biochemistry 2010, 49, 9372-9384.

29.! Lifson, S.; Roig, A. On the theory of helix - coil transition in polypeptides. J.

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30.! Brennan, R. G.; Matthews, B. W. The helix-turn-helix DNA-binding motif. J.

Biol. Chem. 1989, 264, 1903-1906.

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33.! Pauling, L.; Corey, R. B. Compound helical configurations of polypeptide chains:

structure of proteins of the alpha-keratin type. Nature 1953, 171, 59-61.

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38.! Grigoryan, G.; Keating, A. E. Structural specificity in coiled-coil interactions. Curr.

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Biochemistry 1997, 36, 12567-12573.

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!

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Chapter 2 Effect of Side Chain Structure of d-Position

Residue on Coiled Coil Stability

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Introduction

Coiled Coils in Biological Systems

Coiled coils are formed by 3-5% of all amino acids in proteins,¹ and can be found in

transcription factors², cytoskeletal³ and contractile⁴ systems, viral envelope proteins,⁵

and other systems.⁶ In particular, transcription factors are an excellent example to

illustrate the importance of coiled coils. Transcription factors are proteins that bind

specific DNA sequences and thereby regulate gene expression.² Among the numerous

transcription factors, bZIP transcription factors constitute an important class of DNA

binding proteins.^{7, 8} bZIP transcription factors achieve specific DNA binding through

the dimerization of the coiled coil motifs.^{7, 8} Such dimerization results in the formation

of various homodimers and heterodimers, which enhance specific binding between the

proteins and DNA, generate a large number of variations for binding of different DNA

sequences, and therefore form a complex regulatory network in a variety of organisms.^7,

8 This suggests an important role for coiled coils in living systems, and studies of this

structural motif should facilitate the understanding of protein functions and the design

of novel proteins.

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GCN4 Coiled Coil Motif

GCN4 is a bZIP transcription factor in yeast. GCN4 contains a basic region and a

coiled coil region.⁹ The former is responsible for DNA recognition, and the latter

promotes dimerization (Figure 2-1).⁹ The crystal structure of the coiled coil region of

GCN4 was first solved in 1991.² This achievement has turned coiled coils, which was

mainly considered to be structural for decades because coiled coils were first recognized

in fibrous proteins, into a popular research topic that has blossomed in the past twenty

years.^{6, 10, 11} There were two significances. First, it showed that coiled coils participate

in crucial interactions such as transcription. Second, the knowledge obtained from the

physical details of the coiled coil structure provided a means to recognize tertiary

structures by the inspection of primary structures, leading to de novo coiled coil design.^6,

10, 11 The coiled coil region of GCN4 is a 33-residue, 4-heptad repeat leucine zipper (see

Chapter 1 for the general introduction of coiled coils). Leucine zipper bears its name

due to the conserved leucine at the d positions. The a positions are taken up by valine,

so that both a and d!positions are occupied by hydrophobic residues. One exception

occurs at the a position in the third heptad, where there is an asparagine.² This buried

polar residue determines the oligomeric state of the coiled coil, making GCN4 a

(30)

!

homodimeric parallel coiled coil.¹²

Figure 2-1. GCN4-DNA complex (protein data bank code 1YSA).

Coiled Coil Sequence and Structure

The sequence of a coiled coil is characterized by the “heptad repeat”, usually

designated abcdefg. A regular α-helical turn is composed of 3.6 residues.¹⁰ However,

this value is lowered to 3.5 for helices in a coiled coil, leading to the heptad repeat every

two turns of a helix.¹⁰ Inspection of coiled coil sequences led to the concept of "peptide

Velcro", which points out three elements for the formation of a specific coiled coil.^{10, 13}

First, the residues at a and d positions, which are located at the interface between a

coiled coil, need to be hydrophobic residues like leucine, valine, or isoleucine, so that

the coiled coil is stabilized through hydrophobics and van der Waals interactions.

Second, the e and g positions, which are located adjacent to the coiled coil interface,

have to be occupied by charge residues such as glutamate or lysine, which form

interhelical electrostatic interactions that also contribute to coiled coil stability. Last, the

1YSA

Page 1 of 1 Printed at 01:27:10 on Thu Apr 12 2012

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remaining b, c, and f positions have to be hydrophilic residues, since these positions are

located at the solvent-accessing face of a coiled coil.

These features are insufficient to conclude the variety of coiled coils observed in

Nature and laboratory. More precisely, the features are insufficient to predict the

oligomerization (dimer, trimer, or tetramer, etc.), pairing specificity (homodimer or

heterodimer), and orientation (parallel or antiparallel). 20% of the residues at the a and

d positions are polar or charged.¹² Buried polar residues affect the oligomeric state of a

coiled coil.10, 12, 14-16 For instance, the substitution of the two buried asparagines of

GCN4 coiled coil to valine resulted in change in oligomeric state from dimers to a

mixture of dimers and trimers.^{17, 18} These asparagines confer the dimer state through

interhelical hydrogen bonds between the Asn side chains.^{2, 10, 17} Besides, the structure of

the a and d residues also have affect on oligomerization.¹⁸ GCN4 coiled coil mutants,

with the 4 a residues replaced with isoleucine, valine, or leucine yielded dimers,

mixture of dimers and trimers, and trimers, respectively.¹⁸ This switch between

oligomeric states is a consequence of the packing interactions at the coiled coil

interface.¹⁸

Orientation and pairing specificity are greatly influenced by the electrostatic

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!

interactions between the e and g residues.^{10, 19} For instance, placing only negatively

charged residue at the e and g positions in one strand of a coiled coil and only positively

charged residues at the e' and g' positions in another strand, favors the formation of a

heterodimer due to attractive interhelical Coulombic interactions.¹³ Also, deliberate

positioning of oppositely charged residues at g and g' led to the preference for the

antiparallel orientation as a result of the repulsive Coulombic interactions that would be

encountered if the coiled coil was parallel.²⁰

There are still many undiscovered underlying rules for the structures of coiled coils.

Amazing examples include a GCN4 variant with all e positions mutated to valine gave a

parallel tetramer,²¹ whereas another GCN4 variant with all g residues substituted with

either valine or alanine resulted in an antiparallel tetramer.²¹ Still, when all e and g

residues of GCN4 were replaced with alanine, the coiled coil turned out to be a parallel

heptamer.²¹ Predicting the oligomeric state, pairing specificity, and orientation remains

an intriguing challenge.

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Knobs-Into-Holes Interactions of a Dimeric Coiled Coil

Knobs-into-holes (KIH) interactions of a dimeric coiled coil was first described by

Crick in 1953.²² KIH states that a side chain from one helix, referred to as the knob, fits

into the hole surrounded by the side chains of the partner helix.²² For instance, in a

parallel, dimeric coiled coil, the a knob fits into a hole made by d', a', d', and g' of the

neighboring helix, whereas the d knob fits into a hole made by a', e', a', and d' residues

(Figure 2-2a and b).^{2, 11, 18} Furthermore, the a knob projects out of the interface; the

Cα-Cβ vector of the knob is parallel to the Cα-Cα vector at the bottom of the recipient

hole.^{2, 11, 18} The d knob projects directly into the interface; the Cα-Cβ vector of the knob

is perpendicular to the Cα-Cα vector at the bottom of the hole (Figure 2-2c and d).^{2, 11,}

18

Coiled Coil Stability

Many factors contribute to the stability of a coiled coil, such as the coiled coil

propensities of the constituting amino acids,²³ hydrophobic interactions,10, 12, 18, 24, 25

packing of the core residues,^{18, 26-30} and electrostatic interactions.^{10, 31, 32} Herein, coiled

coil propensities, hydrophobic interactions, and side chain packing will be discussed.

Amino acids have different conformational preferences that influence the stability of

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!

(a) (b)

(c) (d)

Figure 2-2. Illustration of knobs-into-hole interactions, parallel packing and

perpendicular packing. (a) Cartoons showing knobs-into-holes interactions. On the left hand side are the projections of each helix strand in a coiled coil onto flat surfaces. On the right is the overlap of the two sheets which reflects the interdigitation of side chains in coiled coil. The angle between the two sheets is the angle between the two strands of helices, which is ~20°. (Reprinted from Woolfson et al. New currency for old rope:

from coiled-coil assemblies to α-helical barrels. Curr. Opin. Struct. Biol. 2012,

doi:10.1016/j.sbi.2012.03.002, with permission from Elsevier.) (b) KIH interactions in GCN4 coiled coil. On the left shows the a knob and the corresponding d'a'd'g' hole, the other is the d knob and the a'e'a'd' hole. (c) One heptad of GCN4 coiled coil. The a knob projects out of the interface, whereas the d knob projects into the interface. (d) Cartoons showing the paralle and perpendicular packing of the a and d knobs. Open circles represent Cα atoms, and solid circles represent Cβ atoms. (GCN4 PDB ID:

2ZTA.)

4 Engineeringanddesign COSTBI-976;NO.OFPAGES10

Figure2

(a)

b

b b b b b b

b e

a

a a

a

d a

d

d d d d d d d d d d d d d d d d d d d d d d d

d c

c

c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c c

c c

g g

g gggggggg g

g g g g g g g g g

g g g g g g g g g e e e

e e

b

b b

b b b b b e

e e e e e e e e e e e e e e e e e e

e a

a

a a a a a a a a

a d

d

a

g d

d d

d d d d d d d d d

d d

c

c c c c c c c c c c c c c c c c

c g

g g e

e

(d)

Type N (xHxxHxx)

Type I (HHxxHxx)

Type II (HHxxHHxx)

Type III (xHxHHxH)

(b) (c)

a = parallel

d = parallel a,d = acute

d = perpendicular

a = perpendicular

c g

f

b e

b f

e c

a d

g

51º

d a

c g

f

b e

d d

b

a e

g

c f

a

c g

b e

f c

103º

154º

a d

g e b

a

d g

f c f

b e

f d

c g

a a

e b

f c g

g d a

e c

f b d

d a

a e

d b

f g c

Current Opinion in Structural Biology

Knobs-into-holes(KIH)interactions.(a)Crick’soriginalhelical-netconcept,whichledtotheideaofKIHpacking.Ontheleft-handside,theheptad repeatsfortwohelices(colouredredandblue)areprojectedontoflatsurfaces.Theseillustratehowthehydrophobica/dseamswindaroundthe helicesintheoppositesensetothatofaregular,right-handeda-helix.Ontheright-handside,thetwonetsareinterlacedtoshowhoweachresidueon

CurrentOpinioninStructuralBiology2012,22:1–10 www.sciencedirect.com

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protein secondary and tertiary structure.^{23, 33} "Helix propensity" describes the

probability of an amino acid to be in a helical state.³⁴ Many studies use monomeric

model helices to measure helix propensities.²³ O'Neil et al. employed a

parallel, homodimeric coiled coil instead.²³ They claimed that a monodimeric helix may

may not be representative of helices found in a protein because a helix in a protein

experiences non-uniform solvent accessibility and dielectric constant.²³ The guest site,

which was substituted with the 20 natural amino acids, was situated at the f position of

the middle heptad. The f position is distant from the dimerization surface, therefore

interhelical packing interactions are avoided. Residues near the guest site were

designated to be small and neutral in order to exclude possible interactions between the

guest residue and nearby residues. As such, differences in coiled coil stabilities solely

depended on the different preferences for the coiled coil conformation of each guest

residue. In other words, this system measures the coiled coil propensity. The result

showed an overall good correlation with data obtained from various monomeric helix

models.²³

Hydrophobic interactions arise from the exclusion of surrounding orderly-arranged

water molecules when hydrophobic moieties approach one another.³⁵ The release of

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!

water molecules leads to the increase in entropy and decrease in free energy.³⁵

Hydrophobicity is quantified by measuring the partition coefficient of a solute between

water and a non-polar solvent, which is further transformed into transfer free energy.³⁵

Hydrophobic interaction is one of the main determinants for coiled coil dimerizaiton.¹⁰

A great deal of work has shown that decrease in the hydrophobicity of the coiled coil

interface decreases coiled coil stability. For instance, the substitution of the buried

asparagine in GCN4 to valine increases the melting temperature (the temperature at

which 50% of the coiled coils are unfolded) by 40°C.¹⁸ The gain in stability is at the

expense of the specificity of oligomeric state (vide supra).^{10, 18} In another study, the

a-position valines and d-position leucines in GCN4 were replaced with asparagine,

threonine, serine, and glutamine, and all of the mutants exhibited lower melting

temperatures.¹² Instead of substituting with polar entities, residues with higher

hydrophobicity were also employed, and coiled coil stability was enhanced. When the

d-position leucines in coiled coil A1 were replaced with trifluoroleucine or

hexafluoroleucine (Figure 2-3), the melting temperatures increased by 10 °C and 22 °C

respectively. The increase in thermal stability was a consequence of the superior

hydrophobicity of trifluoromethyl over methyl groups.^{24, 25}

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Figure 2-3. Structures of leucine, trifluoroleucine and hexafluoroleucine.

The hydrophobic effect is generally agreed to be one of the major determinant factors

in protein folding and stability,^{26, 35} but packing of the residue side chains also play a

significant role in protein stability.^{26, 30} In a typical experiment, a hydrophobic residue

in the core of a protein was substituted with a smaller hydrophobic residue (for example,

leucine to alanine), and the difference between the folding free energies of both the

mutant and wild-type protein was measured.²⁶ This energy difference reflected the

different stabilities of the mutant and wild-type protein.²⁶ Studies revealed that the

energy difference was larger than what would be expected for the transfer free energy,

which is a measure of hydrophobicity.²⁶ The change in protein stability, in this case,

was not caused by hydrophobicity alone, and the discrepancy can be explained by the

difference in packing efficiency.^{26, 27} The formation of cavities within a protein is

destabilizing.^{26, 30} In coiled coils, the geometric properties of buried amino acids

influenced the overall structure and stability.^{18, 27} For example, substituting the 5^th

H₂N OH O

H₂N OH O CF₃

H₂N OH O CF₃ F₃C

Leucine Trifluoroleucine (2S, 4R)-Tfl

Hexafluoroleucine (Hfl)

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!

isoleucine and valine lowers the thermal stability, even though these amino acids are of

similar sizes.²⁷ Leucine is the most favored amino acid at the d position, and is nearly 3

kcal/mole more stabilizing than the similarly sized amino acid isoleucine.²⁷ β-Branched

amino acids are conformationally constrained in α-helices.²⁷ Molecular modeling

indicates that at d positions, the favored rotamers of isoleucine and valine, which are

both β-branched, produce a steric clash between the two amino acids on opposite

helices.²⁷ Recent studies from Gellman's group shows that a'-a-a' vertical interactions

are more prominent than d'-d-d' in antiparallel dimeric coiled coil.^{28, 29} Coiled coil

database mining reveals that the Cα-Cβ vectors of a and a' residues points together and

their trajectories appear to cross, leading to steric repulsion between side chains.²⁹ On

the other hand, Cα-Cβ vectors of d and d' residues are nearly parallel but offset, and the

steric repulsion is avoided.²⁹

Chapter Overview

Coiled coil stability has been intensively investigated over the past two decades, and

the hydrophobicity at the coiled coil interface is the most recognized factor that

determines the stability of this structural motif. Although packing interactions are

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known to affect the stability of proteins and coiled coils, little has been carried out to

probe the relationship between packing and stability. Furthermore, most of these studies

only employed natural amino acids. To further elucidate the relationship between

hydrophobicity, side chain packing, and stability, both natural and non-natural amino

acids with various side chain structures and hydrophobicities were introduced into two

coiled coil systems.

Results and Discussions

Design of GCN4-Derived Peptides

The GCN4 coiled coil region is a well-studied model system for coiled coils,

therefore we chose to study the effect of altering the residues at the d position in this

particular system. There are 4 a positions and 4 d positions in a GCN4 coiled coil

(Figure 2-4a). Initially, all 4 d residues were mutated simultaneously (previously

performed by Hsien-Po Chiu in our lab), but some of these mutants exhibited no coiled

coil conformation by circular dichroism (CD) spectroscopy. This was probably due to

the profound changes in the interface constituents. Therefore, mutation sites were

narrowed down to the central 2 d positions. Still, the second-generation mutant did not

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!

exhibit a coiled coil conformation (Figure 2-5). To enable significant formation of the

coiled coil conformation, only one of the two central d sites (positions 12 and 19) could

be mutated. The GCN4 coiled coil crystal structure showed that both sites were

positioned at the same distance from the center of the coiled coil (Figure 2-4b). In

addition, the side chain of a residue in an α!helix points towards the N-terminus. For

position 12 (Leu12), the side chain points towards position 5, which is a valine at an a

position. Position 19 (Leu19) points towards position 16, which is an asparagine at an a

position. Therefore, position 12 was chosen as the mutation site to exclude the

unfavorable interactions between aliphatic Leu19 and polar Asn16 (Figure 2-4c).

Sixteen different amino acids were incorporated individually at the mutation site (Figure

2-6). These amino acids can be divided into linear (Abu, aminobutyric acid; Nva,

norvaline; and Nle, norleucine), β-branched (Ile, isoleucine; Allo Ile, alloisoleucine; and

Val, valine), γ-branched (Leu, leucine; Tba, tert-butylalanine; Cpa, cyclypentylalanine;

Cha, cyclohexylalanine; Phe, phenylalanine; and Pff, pentafluorophenylalanine), and

polar (Asp, aspartic acid; Asn, asparagine; Glu, glutamic acid; Gln, glutamine).

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(a)

(b) (c)

Figure 2-4. The sequence and the helical wheel diagram of GCN4. (a) Sequence of GCN4. The a and d positions are labeled. The d positions are numbered. (b) Ribbon representation of GCN4 with d-position leucines in space filling. Yellow: Leu12, blue:

Leu19. (c) Helical wheel diagram of GCN4. X denotes the mutation site.

Figure 2-5. CD spectrum of GCN4-Phe2. The central 2 d positions of GCN4-Phe2 were substituted with Phe.

Ac-RMKQ LEDKVEE LLSKNYH LENEVAR LKKLVGE R - OH

d a d a d a d a

5 12 19 26

2ZTA (GCN4 O'Shea d colored)

Page 1 of 1 Printed at 03:38:14 on Wed Apr 18 2012

a b

c

d

e f

g

f’

d’

b’

g’ c’

a’

e’

M V N V V E L E K K E Y A G KNSD

E R H E Q

L E K K

L L X L

L X L L

K K E L

Q E H R E V

V N V M

K E L E

G A Y E K

DSNK

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!

Figure 2-6. Chemical structures of the amino acids.

Peptide Synthesis of GCN4-Derived Peptides

Peptides were synthesized using commercially available reagents by Fmoc-based

solid peptide synthesis (SPPS). However, Allo Ile, Nle, and Cpa were purchased in the

unprotected form, and the Fmoc group had to be incorporated for SPPS (Scheme 1).

The amino acid was dissolved in Na2CO3 solution. This aqueous solution was kept at

pH 10 over ice-water bath. A solution of Fmoc-OSu in tetrahydrofuran (THF) was

added dropwise into the amino acid solution, and the mixture was stirred for 3.5 hours.

For work up, the THF in the mixture was first removed under reduced pressure. The

remaining basic aqueous solution was extracted with diethyl ether. The aqueous fraction

+H₃N O^-

O Abu

+H₃N O^-

O Nva

+H3N O^-

O Nle

O^- O Ile

+H₃N O^-

O Allo Ile

+H₃N O^-

O Val

+H₃N O^-

O Tba

+H₃N O^-

O Cpa

+H₃N O^-

O Cha

+H₃N O^-

O Phe

+H₃N O^-

O Asp O^- O

+H3N O^-

O Asn NH₂ O

+H₃N O^-

O Glu O O^-

+H3N O^-

O Gln O NH₂

+H₃N O^-

O Pff

+H₃N O^-

O Leu

+H₃N

F₅

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was then acidified with 1 M HCl until pH 2, followed by extraction with ethyl acetate.

The organic fraction was dried over Na2SO4, and concentrated in vacuo. The crude was

purified by flash chromatography.

Scheme 1. Incorporation of Fmoc Onto Amino Acid

The identity of the peptides was confirmed by matrix-assisted laser desorption

ionization – time of flight (MALDI-TOF) mass spectrometry. Upon confirmation of the

peptides, purification was carried out on reverse phase high performance liquid

chromatography (RP-HPLC) to greater than 95% purity. The crude yield of peptide

synthesis, molecular formula, calculated [MH⁺], and observed m/z of GCN4 peptides

are shown in Table 2-1.

H₂N O

OH

R Fmoc-OSu, THF

Na₂CO_{3 (aq)}, ice bath, 3.5 hrs

R= Allo Ile Cpa Nle

Fmoc-Allo Ile-OH, 90.1% yield Fmoc-Cpa-OH, 86.9% yield Fmoc-Nle-OH, 78.5% yield

O N

H O

OH O R

d位置胺基酸的側鏈結構對於雙螺旋穩定度的影響

National Taiwan University Master Thesis

Effect of Side Chain Structure of d-Position Residue on Coiled Coil Stability

YI-JEN WENG

Advisor: Richard Ping Cheng, Ph.D.

January, 2013

Abstract

Table of Contents

List of Figures

List of Tables

Abbreviations

Chapter 1 Introduction

X H Y

δ

δ

δ

Chapter 2 Effect of Side Chain Structure of d-Position

Residue on Coiled Coil Stability