fluoroalcohols as structure modiﬁers in peptides and a. kumarmbu.iisc.ernet.in/~pbgrp/305.pdf ·...

S. BhattacharjyaJ. VenkatramanA. KumarP. Balaram

Authors' af®liations:

S. Bhattacharjya, J. Venkatraman and P.

Balaram, Molecular Biophysics Unit, Indian

Institute of Science, Bangalore 560012, India.

A. Kumar, Department of Physics, Indian

Institute of Science, Bangalore 560012, India.

Correspondence to:

Prof. P. Balaram

Molecular Biophysics Unit

Indian Institute of Science

Bangalore 560012, India

Fax: 91 80 3341683

E-mail: [email protected]

Dates:

Received 3 December 1997

Revised 12 December 1998

Accepted 21 January 1999

To cite this article:

Bhattacharjya, S., Venkatraman, J., Kumar, A. &

Balaram, P. Fluoroalcohols as structure modi®ers in

peptides and proteins: hexa¯uoroacetone hydrate

stabilizes a helical conformation of melittin at low pH.

J. Peptide Res., 1999, 54, 100±111.

Copyright Munksgaard International Publishers Ltd, 1999

ISSN 1397±002X

Fluoroalcohols as structuremodi®ers in peptides and

proteins: hexa¯uoroacetonehydrate stabilizes a helicalconformation of melittin atlow pH

Structure stabilization in short peptides by ¯uoroalcohols

has been studied for more than three decades (1±3). Addition

of ¯uoroalcohols such as 2,2,2, tri¯uoroethanol (TFE) to

aqueous solutions of peptides induces a structural transition

from largely random coil conformations to a stable helical

Key words: ¯uoroalcohols; helix stabilization;

hexa¯uoroacetone; melittin; speci®c solvation

Abstract: The effect of hexa¯uoroacetone hydrate (HFA) on the

structure of the honey bee venom peptide melittin has been

investigated. In aqueous solution at low pH melittin is

predominantly unstructured. Addition of HFA at pH < 2.0

induces a structural transition from the unstructured state to a

predominantly helical conformation as suggested by intense

diagnostic far UV CD bands. The structural transition is highly

cooperative and complete at 3.6 M (50% v/v) HFA. A similar

structural transition is also observed in 2,2,2 tri¯uoroethanol

which is complete only at a cosolvent concentration of < 8 M.

Temperature dependent CD experiments support a `cold

denaturation' of melittin at low concentrations of HFA,

suggesting that selective solvation of peptide by HFA is

mediated by hydrophobic interactions. NMR studies in 3.6 M HFA

establish a well-de®ned helical structure of melittin at low pH,

as suggested by the presence of strong NHi/NHi+1 NOEs

throughout the sequence, along with many medium range

helical NOEs. Structure calculations using NOE-driven distance

constraints reveal a well-ordered helical fold with a relatively

¯exible segment around residues T10±G11±T12. The helical

structure of melittin obtained at 3.6 M HFA at low pH is similar

to those determined in methanolic solution and perdeuterated

dodecylphosphocholine micelles. HFA as a cosolvent facilitates

helix formation even in the highly charged C-terminal segment.

100

state (4±7). The induction of helical structure is primarily

sequence-dependent (8±10). Stabilization of other secondary

structures such as b-hairpins or b-sheets is also documented,

albeit to a lesser extent (11, 12). Structural studies of

synthetic protein fragments in aqueous ¯uoroalcohols

provide valuable insights regarding the early stages of

protein folding (5, 13). Stabilization of partly folded states

or `molten globule-like' states of proteins in ¯uoroalcohol/

water mixtures has emerged as a viable strategy to obtain

folding intermediates under equilibrium conditions (14, 15).

The formation of stable secondary structures in aqueous

solution is unfavourable due to the solvation of the peptide

backbone by water, which competes for hydrogen bonding

sites and disrupts intramolecular interactions (16). Unlike in

the folding of proteins, peptide fragments are generally too

short in length to nucleate secondary structures within an

initial globular state formed by `hydrophobic collapse'.

Recently we have shown that aqueous hexa¯uoroacetone

(HFA), like TFE, can be used as a structure stabilizer for

peptide sequences from proteins, with HFA displaying

superior structure stabilizing properties as compared to

TFE (17). On the basis of our results we have proposed a

model for interactions of ¯uoroalcohols with the peptides (7,

17). The model suggests direct interactions of ¯uoroalcohols

with peptides, driven primarily by hydrophobic interactions

involving the tri¯uoromethyl groups of HFA. As part of a

programme to systematically investigate the effect of HFA

on different peptide sequences, we have studied the

conformation of melittin in aqueous HFA solution at low

pH. Melittin, a cytolytic membrane active peptide from

honey bee venom, consists of 26 amino acid residues

(GIGAVLKVLTTGLPALISWIKRKRQQ) and is amidated at

the C-terminus. The conformations of melittin have been

extensively investigated in crystals by X-ray diffraction (18)

and under various solution conditions (19±24). A tetrameric

helical conformation of melittin has been established by

crystallography and in high salt and high pH solutions (18,

19). However, the conformation is largely random coil in

aqueous solution at low pH due to unfavourable charge/

charge repulsion in the C terminus KRKR segment (25). A

monomeric helical structure is also reported in the presence

of lipids and in membrane mimetic environments (21, 23,

26). In this report, we demonstrate a helical conformation of

melittin at low pH in the presence of aqueous HFA by CD

and NMR. Our results show a sharp transition from a

random coil conformation to a largely helical conformation

on titrating an aqueous solution of melittin with HFA. The

transition is completed by 3.6 m HFA (50% v/v). NMR

studies performed at 3.6 m HFA, pH 2.0, establish a stable

helical conformation. Thermal melting experiments mon-

itoring CD bands, performed at different concentrations of

HFA support a `cold denaturation' of the helical structure,

suggesting hydrophobically driven solvation of the peptide

by ¯uoroalcohols. Comparative studies in aqueous TFE and

HFA show that the latter is a superior structure stabilizer.

Materials and Methods

Melittin was obtained from Sigma Chemical Co. The purity

of the material was checked by a reverse phase HPLC (C18,

5m) using methanol±water gradients (70±95%) containing

1% tri¯uoroacetic acid. The hexa¯uoroacetone trihydrate

(HFA, a covalent hydrate of the ketone, a gem diol,

hexa¯uoropropan-2,2-diol) was obtained from Aldrich Che-

mical Co.

Circular dichroism (CD)

All CD spectra were recorded on a JASCO-J-500 A spectro-

polarimeter using cells of path length 1 or 2 mm. Peptide

concentrations were determined using the single tryptophan

absorbance at 280 nm (e280 = 5600 m/cm). The peptide

concentrations for CD measurements range from 25 mm to

40 mm. The pH of the samples were adjusted to < 2 with

0.1 N HCl, after addition of HFA. Band intensities are

expressed as molar ellipticities [h]M, deg. cm2/dmol. The

molar concentrations of HFA trihydrate and TFE, assuming

that volumes are additive on mixing, were calculated in the

following manner: moles of HFA = (1.59 3 V)/220; moles of

TFE = (1.38 3 V)/100; moles of water in HFA solution =

[(36/220 3 1.59 3 V + (1000 - V)]/18; moles of water in TFE

solution = (1000 - V)/18, where V is the volume of HFA or

TFE in 1000 mL of solution, 1.59, 1.38 are the density of

HFA trihydate and TFE in g/cm3, respectively, Mr

(H2O) = 18, Mr (HFA, trihydrate) = 220 and Mr

(TFE) = 100. The neat trihydrate is considered as one

molecule of the diol and two molecules of water.

Fluorescence

Fluorescence spectra were recorded on a Hitachi 650-60

spectro¯uorimeter using cells of path-length 1 cm. Excita-

tion and emission band passes were set at 5 nm.

Bhattacharjya et al . Melittin helix in hexa¯uoroacetone

J. Peptide Res. 54, 1999 / 100±111 | 101

NMR spectroscopy

1H NMR spectra were recorded on a Bruker AMX 500

spectrometer. The NMR spectra were recorded in water

solution containing 3.6 m HFA (50% v/v) with 10% D2O for

locking. The pH of all samples were adjusted to 2 by 0.1 m

HCl, after addition of HFA. The peptide concentrations

varied from 1 mm to 5 mm. All the 2-D NMR experiments

were carried out at a concentration of 3 mm. A set of

temperature-dependent 1-D experiments were performed to

®x the temperature for the 2-D experiments. 2-D double

quantum ®ltered COSY, TOCSY and NOESY experiments

were performed at different temperatures (298 K, 303 K and

310 K) for complete resonance assignment. NOESY experi-

ments were done at 200 ms and 300 ms mixing times. A

70-ms mixing time was used for the TOCSY experiments.

The residual water was suppressed by presaturating the

water resonance by a 55-db pulse in a recycle delay of 1.5 s.

The chemical shifts are with reference to sodium 3-

(trimethylsilyl)-propionate-d4 (TSP). All the 2-D data were

acquired at 1 K 3 512 data points with 40±72 transients

using a 5500±6000 Hz spectral width. 2-D data were zero

®lled to 1K 3 1K data points, the FIDs were multiplied by

sine p/4 function prior to Fourier transformation.

Determination of NMR constraints

Depending on the cross-peak intensities the NOEs were

classi®ed into three different distance categories: strong,

2.0 AÊ -3.0 AÊ ; medium, 2.5 AÊ ±3.5 AÊ ; and weak, 3.5 AÊ ±4.8 AÊ .

In case of overlap, pseudoatoms were used allowing 1 AÊ

relaxation in distance criteria. All the backbone to back-

bone, backbone to side chain and side chain to side chain

NOEs were considered. A total of 310 distance constraints of

which 195 were intraresidue were used in structure

calculations. The dihedral angles (w) used as constraints

were calculated using an empirical relationship given by

Wishart et al. (27) relating the chemical shift deviation of

the C_H proton from the random coil values (Dd) and the

dihedral angle w, Dd = - 0.013 w±1.20. The dihedral angles y

were allowed to vary in the range of + 1208 to - 408 for all the

residues.

Structure calculation

Structure calculations were performed using Dyana

(Dynamics algorithm for NMR application) version 1.2

(28, 29) on a Sun Workstation. A set of 30 starting structures

with random dihedral angles (w, y) were generated, which

were further submitted for simulated annealing protocols

with NMR restraints. The force constants were ®xed to a

value of 1 or 0.5. The distance constraint violations were

calculated for all the 30 structures. Fifteen structures having

low target functions and least restraints violations were

selected for further analysis. The structures were viewed and

superposed using the insight ii software package running on

a Silicon Graphics Work Station.

Results

CD studies

Figure 1 shows far UV CD spectra of melittin in water, HFA/

water and TFE/water solutions at low pH. The CD spectrum

in aqueous solution is typical of a random coil conformation

showing a negative band at < 200 nm. A dramatic structural

transition from the random coil conformation to a helical

state is observed in presence of HFA, as shown by the

appearance of two intense CD bands at < 208 and 220 nm.

The HFA-induced structural transition is very sharp and is

complete by 3.0±3.8 m HFA. The intensities of the CD bands

obtained in < 6.9 m TFE±water are comparable to that in

3.6 m HFA±water (note that the molar concentrations of

H2O in the two cases are < 27.78 m and 36 m, respectively).

The structural transition in TFE is observed at higher

cosolvent concentrations and is complete only at < 8 m TFE

(Fig. 1, inset), suggesting that HFA is a more potent

structure stabilizer.

Thermal melting of melittin in various concentrations of aqueous

HFA

The temperature dependence of the CD band intensities for

melittin in aqueous HFA are shown in Fig. 2. At an HFA

concentration of 3.6 m, when the structural transition is

essentially complete, the helical conformation is more

populated at low temperature. There is signi®cant decrease

in ellipticity with increasing temperature suggesting of

thermal unfolding. However, at lower concentrations of

HFA (0.16 m), well below the concentration range for

completion of the structural transition, a `cold denaturation'

of melittin is observed. There is a loss of CD ellipticity at

both low and high temperature, with maximum helix

content observed at < 308C. The diminished intensities of

the CD bands at high temperature can be interpreted as

conventional thermal unfolding of the helical structure, due

to breakage of intramolecular hydrogen bonds. The loss of


102 | J. Peptide Res. 54, 1999 / 100±111

far UV CD bands at low temperature can only be

rationalized by invoking diminished hydrophobic interac-

tions which stabilize the helical fold. The diminution of

hydrophobic interaction at low temperature is well known

for folded globular proteins which are mainly stabilized by

hydrophobic non-polar side chain/side chain interactions

inside the protein core. The forces responsible for the

stabilization of secondary structures are mainly hydrogen

bonds or salt bridges. Therefore, secondary structures are

indeed more stable at low temperature. Proteins should in

principle exhibit unfolding transitions at both low and high

temperatures (30) and indeed several examples of cold

denaturation have been observed (31).

The destabilization of the helical structure of melittin at

low temperature in aqueous HFA is suggestive of a direct

association of the ¯uoroalcohol with the peptide by hydro-

phobic interactions. Recently, we have proposed a model for

the interaction of ¯uoroalcohols with peptides (17). The

model essentially suggests selective solvation of the peptides

by ¯uoroalcohols, displacing surrounding water molecules, a

process driven mainly by hydrophobic interactions involving

¯uoroalkyl group(s) (CF3) of the cosolvents and side chains on

the peptide. The hydrophobic nature of ¯uoroalkyl groups is

evident from the water-repellent properties of the polymer

`Te¯on' (32) and the dramatically lower critical micelle

concentrations of ¯uorosurfactants (33, 34). Fluoroalcohols

provide a `Te¯on-like' hydrophobic coat to peptide solutes,

promoting intramolecular hydrogen bond formation. The

cold denaturation effect observed in melittin at concentra-

tions of HFA below that required for the structural transition

supports hydrophobic association of ¯uoroalcohols with the

peptide through CF3 groups. At higher concentrations of HFA

cold denaturation is not observed, presumably due to the

saturation of all the sites available for binding HFA. It should

benotedthat inastudybyRamalingamandBello (20) the `cold

denaturation' as manifested by loss of CD ellipticities at low

temperaturehas indeedbeenobserved formelittinderivatives

containing alkanoyl substitutents on the Lys sidechains. For

Figure 2. Temperature dependence of CD ellipticity at 222 nm for

melittin at 0.14 m HFA, 0.36 m HFA and (inset) 3.6 m HFA at pH 2.0. The

peptide concentrations were 20 mm, and a cell of 2 mm path-length was

used.

Figure 1. Left: far UV CD spectra of melittin

in aqueous solution and in various

concentrations of aqueous HFA and aqueous

TFE at pH 2.0. Right: the dependence of

molar ellipticity values at 222 nm [h]M on

HFA concentrations. Inset: the dependence of

molar ellipticity at 222 nm on TFE

concentrations. The peptide concentrations

were 40 mm, and a cell of 1 mm path-length

was used for all the experiments.


J. Peptide Res. 54, 1999 / 100±111 | 103

native melittin itself cold denaturation is not observed at

acidic pH. These observations relate to structural transitions

involving the tetrameric aggregate which is stabilized by

hydrophobic interactions.Similarobservations ofa lossofCD

ellipticity at low temperature have also been made for a

synthetic helix forming peptide at low concentrations of

hexa¯uoroisopropanol (35).

Aggregation state of melittin in aqueous HFA

Circular dichroism studies were carried out across a range of

melittin concentrations (10±100 mm) in 3% HFA/H2O

solutions, pH 2.0. The CD spectra were invariant across

the concentration range studied suggesting that both the CD

as well as the temperature dependence of the CD (data not

shown) are independent of melittin concentration. Fluores-

cence emission spectra of 40 mm melittin in 3% HFA,

pH 2.0 showed an emission maximum of 356 nm. This is

suggestive of a monomeric melittin species with completely

solvent-exposed tryptophan (36). Attempts to tetramerize

this putative melittin monomer by the addition of phos-

phate salt (36) proved successful, with the ¯uorescence

emission maximum progressively blue shifting on addition

of phosphate (Fig. 3). At a phosphate concentration of 0.3 m,

the observed ¯uorescence maximum is 344 nm. Fluoroal-

cohols like TFE have been shown to quench tryptophan

¯uoresence in proteins (10, 37, 38). Quenching is probably a

consequence of direct interaction of the ¯uoroalcohol with

aromatic rings. HFA has also been shown to be an effective

quencher of tryptophan ¯uorescence (R. Rajan & P. Balaram,

unpublished data). Therefore, quenching experiments were

undertaken on preformed tetramer and monomer forms of

melittin. Successive addition of HFA to the melittin

tetrameric and monomeric forms resulted in quenching in

both cases, with the ¯uorescence emission maximum in the

case of tetrameric melittin progressively red shifting

simultaneously. This is suggestive of disassociation of the

melittin tetramer accompanying quenching on addition of

HFA. Surprisingly, Stern±Volmer plots (Fig. 4) appear to

indicate that the quenching is more effective in the case of

tetrameric melittin as compared to monomeric melittin.

This could perhaps be interpreted as a selective trapping of

HFA within the unravelling melittin tetramer, the enhanced

local concentration of quencher leading to greater trypto-

phan quenching. As expected, control experiments with KI

as quencher show the quenching of tryptophan ¯uorescence

Figure 3. Dependence of the ¯uorescence spectra of melittin in 3%

HFA, pH 2.0 on increasing concentrations of phosphate salt. The peptide

concentration was 40 mm, the salt used was potassium dihydrogen

phosphate (KH2PO4) and a cell of path-length 1 cm was used for all

¯uorescence experiments.

Figure 4. Differential quenching by HFA of the tetrameric and

monomeric forms of melittin in aqueous solutions. The peptide

concentrations were 40 mm. Tetramerization of melittin was achieved

by the addition of 0.7 m KH2PO4 prior to quenching experiments.

Peptide concentrations were continually adjusted to 40 mm to

compensate for the increase in volume on addition of HFA. Inset:

quenching of the tetrameric and monomeric forms of melittin by KI.


104 | J. Peptide Res. 54, 1999 / 100±111

Figure 5. Partial TOCSY spectrum (CaH±

NH) of melittin in 3.6 m HFA, pH 2.0 at

313 K indicating amino acid spin systems.

Peptide concentration was 3 mm.

Figure 6. Partial NOESY spectrum of

melittin in 3.6 m HFA, pH 2.0 at 305 K

showing NH/NH NOE connectivities. A

mixing time of 300 ms was used for the

NOESY experiment.


J. Peptide Res. 54, 1999 / 100±111 | 105

in monomeric melittin is greater than that observed in the

tetrameric form (inset, Fig. 4).

NMR studies

Sequence-speci®c resonance assignments

All NMR experiments were performed in 3.6 m HFA/water

mixtures since CD spectra indicate that the structural

transition from random coil to a helical state is complete at

that solvent composition. Sequence-speci®c resonance

assignments were achieved by standard procedures (39),

using TOCSY, double quantum ®ltered COSY and NOESY

experiments. NMR experiments were performed at different

temperatures to overcome resonance overlap. Spin systems of

the amino acids are identi®ed in a TOCSY spectrum (Fig. 5),

which are distinguished sequence-speci®cally with the help

of many short range (aNi,i, aNi,i+1, NHi/NHi) and medium

range (aNi,i+3, aNi,i+4 and abi,i+3) NOEs (Figs 6, 7). The

chemical shift values of the amino acids are listed in Table 1.

Chemical shift analysis

CaH proton chemical shifts in peptides and proteins are

highly conformation sensitive (27). In a stable helical

conformation CaH protons show a signi®cant up®eld shift

from the random coil values (39), whereas a down®eld shift

is characteristic of well de®ned b-sheet structure (27, 40).

Most of the CaH protons of melittin in aqueous HFA

solution (3.6 m) experience a remarkable up®eld shift

(Fig. 8). Interestingly, the deviation of CaH from the

random coil are considerably reduced for the central residues

T10±T11±G12±L13, indicating some conformational ¯ex-

ibility around that segment. The crystal structure of

tetrameric melittin (2 AÊ ) indeed suggests a deviation from

an ideal helical conformation around residues 11±12 (18).

Structural distortions with enhanced conformational ¯ex-

ibility in the centre of the melittin molecule are also

suggested by the amide proton exchange rates in CD3OD;

the exchange rates were retarded in an analogue containing

Ala in place of Pro at residue 14 (41, 42).

Nuclear Overhauser effects (NOE)

Figure 6 represents NOEs between the amide protons.

Presence of intense sequential NH/NH NOEs along with

many medium to weak NH/NH (i + 2, i + 3) NOEs suggests

a helical conformation. The helical conformation is also

strongly supported by intense intraresidue aN, weak to

medium sequential aN NOEs along with medium range aN

(i, i + 3, i + 4) and ab (i, i + 3) NOEs (Figs 7, 9). The

Figure 7. Partial NOESY spectrum of

melittin in 3.6 m HFA, pH 2.0 at 305 K

showing CaH/NH NOE connectives. A

mixing time of 300 ms was used for the

NOESY experiment.


106 | J. Peptide Res. 54, 1999 / 100±111

sequential NH/NH NOEs start from residue 2 and propagate

up to residue 25, encompassing the ¯exible segment T10±

T11±G12. The observation of NOEs between L13

NH«dCH2 P14 and P14 dCH2«NH A15 suggest that the

helical conformation has extended across the Pro residue. It

may be noted that Pro can be accommodated into polypep-

tide helices with only marginal distortion despite the loss of

an internal hydrogen bond (43). The helical conformation of

melittin stabilized by aqueous HFA appears to be mono-

meric since concentration dependence experiments do not

show any change in line width or chemical shifts (data not

shown). Moreover, the diagnostic long-range NOEs between

Ile 2 dCH3 and Trp 19 indole proton, which was observed in

the aggregated states of melittin (23), was not detected in the

present study, supporting existence of a monomeric helical

structure. The peptide bond around L13 and P14 adopts a

trans conformation as evident from strong NOEs between

L13 CaH«dCH2 P14 and L13 NH«dCH2 of P14, as opposed

to conformations in aqueous solution at low pH where both

the cis and trans conformations coexist (25).

Description of the structure

The observed pattern of NOEs supports a helical con-

formation of melittin in 3.6 m HFA at pH 2.0. Figure 10

shows the superposition of backbone atoms (Ca, C' and N)

of 15 lowest energy structures along with the average

NMR structure. The ®rst and last four residues exhibit

relatively high RMSD values as compared to the other

residues suggesting somewhat more ¯exible termini, with

a rigid middle segment. However, T11 and G12 show

RMSD values close to the terminal residues. Calculations

of local RMSDs with a segment length of three residues

suggest the highest mobility of this segment (T11, 0.17 AÊ ,

G12, 0.19 AÊ ), in contrast to the other residues (# 0.06 AÊ )

(data not shown). The average w, y-values are in the

allowed region of the Ramachandran map (44) indicating

stereochemical acceptability of the structure. The back-

bone dihedral angles (w, y) (Table 2) are well clustered in

the helical region of the Ramachandran map for all the

residues.

Comparison between crystal and NMR structures

In the crystal structure of melittin the asymmetric unit

consists of two independent chains, both of which adopt a

bent helical structure with the kink occurring at the T11±G12

segment (18). This is presumably a consequence of accom-

modating P14 into the helical structure. Insertion of a Pro

residue (at position n) in the centre of helices requires

accommodation of the dCH2 group in place of the amide

Table 1. Chemical shift (p.p.m.) values for melittin in 50% HFA,pH 2.0, 303K

Residue NH CaH CbH CcHCdH &others

G1Ð 3.98, 4.01

I2 8.38 4.21 1.80 CcH2, 1.6,

1.3, CcH3

0.90

0.90

G3 8.93 3.81

A4 7.67 4.17 1.52

V5 7.28 3.75 2.25 1.08, 1.00

L6 8.00 4.13 1.85 1.55 0.90

K7 8.03 4.01 2.05 1.50 1.70, CeH

3.02, NeH

7.46

V8 7.85 3.74 2.35 1.10, 1.02

L9 8.56 4.19 1.90 1.49 0.89

T10 8.15 4.28 4.42 1.38

T11 7.70 4.40 4.45 1.36

G12 8.10 4.08, 4.18

L13 8.10 4.45 1.88, 1.75 1.05 0.98

P14 Ð 4.38 2.40 2.15 3.80, 3.61

A15 7.33 4.18 1.52

L16 7.79 4.32 2.05, 1.77 1.05 0.93

I17 8.57 3.81 1.98 CcH2 1.34,

1.70, CcH3

0.95

0.85

S18 7.96 4.16 4.02

W14 8.04 4.45 3.49, 3.68 C2H,

7.21, C4H

7.72, C5H

7.12, C6H

7.22, C7H

7.48,

indole NH

9.48

120 8.73 3.51 2.05 1.27 0.91

K21 8.61 3.90 1.98, 1.90 1.40 1.70, CeH

2.98

R22 7.77 4.08 1.96, 180 1.63 3.20, NdH

6.90

K23 8.31 3.98 1.65 1.20 1.48, CeH

2.80, 2.86

R24 8.34 4.15 1.99, 1.85 1.70 3.12,

3.18, NdH

6.65

Q25 7.89 4.27 2.12 2.47

Q26 7.91 4.29 2.20 2.50


J. Peptide Res. 54, 1999 / 100±111 | 107

hydrogen. In addition to loss of a single intramolecular

hydrogen bond, dihedral angle distortion at preceding

residues are necessary to relieve short contacts in an ideal

helix between Pro (n) dCH2 and the C = O group of a residue at

the (n-3) position (45). This distortion is clearly seen at T11

and G12 in both conformers in the crystal. The two

conformers differ in the value of w at T11 (- 1318 for the A-

chain and - 878 for the B-chain). In order to assess the nature of

distortion in the central segment in solution, short inter-

proton distances (# 4 AÊ ) were computed for the central

segment in both conformers. The following distances serve as

useful diagnostics: conformer A; V8 CaH ± T11 NH 2.95 AÊ ,

V8 CaH ± G12 NH 2.98 AÊ . In an ideala-helical conformation,

the corresponding distances are daN (i, i + 3) 3.4 AÊ and daN (i,

i + 4) 4.2 AÊ . For conformer B the distances were, V8 CaH ±

T11 NH 3.61 AÊ , V8 CaH ± G12 NH 3.89 AÊ .

Inspection of Fig. 7 reveals a strong cross-peak which may

be assigned to V8 CaH/T11 NH NOE. It should be noted that

there is overlap between the V8 CaH and one of the W19

bCH2 protons, which is signi®cantly down®eld shifted in

melittin (3.68 p.p.m.). The cross-peak corresponding to the

V8 CaH/G12 NH NOE is appreciably less intense. However,

spectral overlap precludes a clear distinction between the

possible conformers in the central segment. Dynamic

averaging over a limited region of conformational space

must also be considered in interpreting the solution data.

The average structure derived from NMR is much less

distorted in the T10±G12 segment than the crystal structure

(Table 2).

Discussion

Melittin in aqueous solutions at low pH is unstructured

because of the presence of several positively charged groups

at the C terminus. Helix induction is usually achieved at

high pH or by the addition of strong counter ions such as

phosphate. Hexa¯uoroacetone hydrate (HFA) has been

shown in this study to be a very effective helix inducer

even at low pH. CD and ¯uorescence data suggest that the

Figure 8. The CaH chemical shift deviations from the random coil

values for melittin in 3.6 m HFA, 303 K, pH 2.0.

Figure 9. Summary of observed NOEs for melittin in 3.6 m HFA, pH 2.0. The amino acid sequence is shown at the top. The intensities of the NOEs

are categorized as strong, medium or weak and marked accordingly by different shades.


108 | J. Peptide Res. 54, 1999 / 100±111

peptide exists as a monomeric species at HFA concentra-

tions as low as < 3% (v/v). Two-dimensional NMR studies

carried out in 50% aqueous HFA (v/v) ®rmly establish a

helical backbone. The conformation determined for melit-

tin in aqueous HFA at low pH is a continuous helix over

the entire length of the molecule with relatively minor

distortions in the segment T10±T11±G12. A similar

continuous monomeric helix has been established for

melittin in methanol solution at an effective pH of 5.0

(21). A 500-MHz study of melittin in perdeuterated

dodecylphosphocholine micelles also yielded a largely

helical conformation with some evidence of structural

disorder at K23 and R24, where no sequential NOEs were

obtained (26). Interestingly, a transferred NOE study of

melittin in perdeuterated phosphatidylcholine vesicles

yielded two distinct helical segments L6±L10 and L13±

K21, with a less structured T11±G12 segment. The ®ve C-

terminal residues R22±Q26 were found to be unstructured

(22). Two spatially distinct N and C terminal segments of

melittin had been reported in the dodecylphosphocholine

micelles in an earlier investigation (46). Melittin exists as

an unstructured monomer in aqueous solution at neutral

pH, at low salt concentrations; upon addition of counter-

ions, helix formation ensues accompanied by aggregation

to a tetrameric structure (19, 47). Thus in melittin,

secondary structure formation and quaternary association

appear interdependent in aqueous solution. Organic cosol-

vents induce helix formation in melittin, with a recent

study demonstrating the order of effectiveness, hexa¯uor-

oisopropanol . TFE . isopropanol . ethanol . methanol

(24). The absence of a helical conformation in melittin

under conditions where the basic residues are positively

charged has been attributed to electrostatic repulsion,

which presumably destabilizes folded structures in the

monomer and impedes aggregation to form the tetramer.

Charge neutralization by the addition of counterions or

chemical modi®cation of the positively charged side chains

result in helix stabilization (19, 20). In the present study

helix stabilization over the entire length of the molecule

has been demonstrated even under conditions where the

basic residues are protonated. The absence of

Figure 10. Left: superposition of backbone atoms (Ca, C', N) of 15

melittin structures generated using Dyana. Right: the average NMR-

derived structure.

Table 2. Dihedral angles for melittin in crystals(A and B chains) and average NMR structure

Crystal structurea NMR structureb

Residues (deg.) (deg.) (deg.) (deg.)

ILE 2 ±51(±64) ±46(±30) ±79u12 ±43u11

GLY 3 ±63(±70) ±39(±40) ±73u10 ±42u12

ALA 4 ±70(±68) ±47(±45) ±64u7 ±44u5

VAL 5 ±49(±60) ±54(±43) ±61u9 ±41u6

LEU 6 ±57(±61) ±38(±45) ±59u6 ±42u5

LYS 7 ±71(±61) ±35(±39) ±62u7 ±44u8

VAL 8 ±66(±64) ±44(±39) ±56u7 ±41u6

LEU 9 ±59(±57) ±48(±33) ±63u7 ±40u7

THR 10 ±60(±87) ±42(±22) ±64u8 ±40u6

THR 11 ±87(±131) ±36(±42) ±61u9 ±24u8

GLY 12 ±99(±84) ±24(±33) ±71u10 ±27u12

LEU 13 ±62(±62) ±42(±46) ±61u8 ±62u10

PRO 14 ±55(±54) ±46(±46) ±74u7 ±13u10

ALA 15 ±62(±68) ±38(±41) ±82u8 ±39u6

LEU 16 ±62(±58) ±49(±41) ±82u7 ±39u6

ILE 17 ±60(±58) ±46(±49) ±74u6 ±50u6

SER 18 ±59(±60) ±45(±46) ±60u9 ±48u7

TRP 19 ±60(±49) ±51(±59) ±54u9 ±43u8

ILE 20 ±60(±54) ±46(±45) ±60u7 ±44u7

LYS 21 ±56(±59) ±50(±42) ±53u9 ±45u7

ARG 22 ±50(±61) ±58(±45) ±57u7 ±43u6

LYS 23 ±47(±69) ±44(±32) ±57u8 ±45u10

ARG 24 ±64(±71) ±39(±39) ±60u12 ±41u10

GLN 25 ±65(±76) ±18(±22) ±62u13 ±56u12

a. The dihedral angles for the A chain is given inparentheses. From reference 18. b. The dihedral anglesfor the NMR structures are averaged over 15 relatedconformers.


J. Peptide Res. 54, 1999 / 100±111 | 109

concentration-dependent chemical shifts in 1-D NMR

spectra in 3.6 m HFA and the relatively sharp resonances

suggest the absence of aggregated species. While helix

formation over the hydrophobic stretch (1±20) in aqueous

HFA is unsurprising, stabilization of the helical fold even

at the C terminus is intriguing. It is likely that the side

chains of Arg and Lys residues, which contain as many as

three to four polymethylene units, are hydrophobic enough

to necessitate adoption of compact side chain conforma-

tions involving gauche conformations about C±C bonds

minimizing exposure of alkyl chains in water. Such

structures should bring the positively charged groups in

proximity in a helical conformation, resulting in increased

electrostatic repulsion. The balance between folded and

unfolded backbone structures may be in¯uenced by the

nature of the side chain conformations. Addition of a

cosolvent such as HFA should permit selective solvation of

the hydrophobic segments of the side chains, permitting

extended polymethylene conformations which will in turn

minimize electrostatic repulsion. Thus helix formation

may be facilitated even under conditions where the side

chains are protonated. An understanding of the role of side

chain interactions in in¯uencing backbone conformation

may be facilitated by studies of analogues where the basic

segment has been transferred, as in the retro derivatives of

melittin (48). The present study emphasizes the utility of

HFA hydrate as a structure modi®er in peptides and

proteins. Helix stabilization in aqueous media may be

largely mediated by dessication (or desolvation) of the

peptide backbone (7, 17), a feature emphasized in a recent

study of tri¯uoroethanol mediated effects (49). Helix

induction in melittin upon addition of diverse alcoholic

cosolvents has also been recently investigated (50). HFA is a

particularly useful solvent for NMR studies where the

absence of non-exchangeable protons and the higher

viscosity of HFA/H2O mixtures makes it a particularly

attractive system for determination of nuclear Overhauser

effects.

Acknowledgments: NMR studies were carried out at the

National High Field NMR Facility at the Tata Institute of

Fundamental Research, Mumbai. Model building and structure

calculations were carried out at the Sophisticated Instruments

Facility, Indian Institute of Science, Bangalore.

References

1. Goodman, M. & Listowsky, I. (1962)

Conformational aspects of synthetic

polypeptides. VI. Hypochromic spectral

studies of oligo-c-methyl-l-glutamate

peptides. J. Am. Chem. Soc. 84, 3770±3771.

2. Nelson, J.W. & Kallenbach, N.R. (1989)

Persistence of the alpha-helix stop signal in

the S-peptide in tri¯uoroethanol solutions.

Biochemistry 28, 5256±5261.

3. Cammers-Goodwin, A., Allen, T.J., Oslick,

S.L., McClure, K.F., Lee, J.H. & Kemp, D.S.

(1996) Mechanism of stabilization of helical

conformations of polypeptides by water

containing tri¯uoroethanol. J. Am. Chem.

Soc. 118, 3082±3090.

4. Osterhout, J., Baldwin, R.L., York, E.,

Stewart, J.M., Dyson, H.J. & Wright, P.E.

(1989) 1H NMR studies of the solution

conformations of an analogue of the C-

peptide of ribonuclease A. Biochemistry 28,

7059±7064.

5. Bolin, K.A., Pitkeathly, M., Mirankar, A.,

Smith, L.J. & Dobson, C.M. (1996) Insight

into a random coil conformation and an

isolated helix: structural and dynamical

characterisation of the C-helix peptide from

hen lysozyme. J. Mol. Biol. 261, 443±453.

6. Kemmink, J. & Creighton, T.E. (1995) Effects

of tri¯uoroethanol on the conformations of

peptides representing the entire sequence of

bovine pancreatic trypsin inhibitor.


7. Rajan, R. & Balaram, P. (1996) A model for the

interaction of tri¯uoroethanol with peptides

and proteins. Int. J. Peptide Protein Res. 48,

328±336.

8. Sonnichsen, F.D., Van Eyk, J.E., Hodges, R.S.

& Sykes, B.D. (1992) Effect of tri¯uoroethanol

on protein secondary structure: an NMR and

CD study using a synthetic actin peptide.


9. Hamada, D., Kuroda, Y., Tanaka, T. & Goto,

Y. (1995) High helical propensity of the

peptide fragments derived from beta-

lactoglobulin, a predominantly beta-sheet

protein. J. Mol. Biol. 254, 737±746.

10. Shiraki, K., Nishikawa, K. & Goto, Y. (1995)

Tri¯uoroethanol-induced stabilization of the

alpha-helical structure of beta-lactoglobulin:

implication for non-hierarchial protein

folding. J. Mol. Biol. 245, 180±194.

11. Blanco, J.F., Jimenez, M.A., Pineda, A., Rico,

M., Santoro, J. & Nieto, J.L. (1994) NMR

solution structure of the isolated N-terminal

fragment of protein-G B1 domain. Evidence of

tri¯uoroethanol induced native-like beta-

hairpin formation. Biochemistry 33, 6004±

6014.

12. Ramirez-Alvardo, M., Blanco, F.J. & Serrano,

L. (1996) De novo design and structural

analysis of a model beta-hairpin peptide

system. Nature Struct. Biol. 3, 604±612.

13. Dyson, H.J., Merutka, G., Waltho, J.P.,

Lerner, R.A. & Wright, P.E. (1992) Folding of

peptide fragments comprising the complete

sequence of proteins. Models for initiation of

protein folding. I. Myohemerythrin. J. Mol.

Biol. 226, 795±817.

14. Buck, M., Radford, S.E. & Dobson, C.M.

(1993) A partially folded state of hen egg

white lysozyme in tri¯uoroethanol:

structural characterization and implications

for protein folding. Biochemistry 32, 669±678.

15. Bhattacharjya, S. & Balaram, P. (1997)

Hexa¯uoroacetone hydrate as a structure

modi®er in proteins: characterization of a

molten globule state of hen egg-white

lysozyme. Protein Sci. 6, 1065±1073.


110 | J. Peptide Res. 54, 1999 / 100±111

16. Scholtz, J.M. & Baldwin, R.L. (1992) The

Mechanism of Alpha-Helix Formation by

Peptides. Annu. Rev. Biophys. Biomol.

Struct. 21, 95±118.

17. Rajan, R., Awasthi, S.K., Bhattacharjya, S. &

Balaram, P. (1997) `Te¯on-coated peptides':

hexa¯uoroacetone trihydrate as a structure

stabilizer for peptides. Biopolymers 42, 125±

128.

18. Terwilliger, T.C. & Eisenberg, D. (1982) The

structure of melittin. I. Structure

determination and partial re®nement. J. Biol.

Chem. 257, 6010±6022.

19. Bello, J., Bello, H.R. & Granados, E. (1982)

Conformation and aggregation of melittin:

dependence on pH and concentration.


20. Ramalingam, K. & Bello, J. (1993)

Conformational and aggregational states of

omega-aminoacylmelittin derivatives.


21. Bazzo, R., Tappin, M.J., Pastore, A., Carver,

J.A. & Campbell, I.D. (1988) The structure of

melittin. A 1H-NMR study in methanol. Eur.

J. Biochem. 173, 139±146.

22. Okada, A., Wakamatsu, K., Miyazawa, T. &

Higashijima, T. (1994) Vesicle- bound

conformation of melittin: transferred nuclear

Overhauser enhancement analysis in the

presence of perdeuterated

phosphatidylcholine vesicles. Biochemistry

33, 9438±9446.

23. Brown, L.R., Lauterwein, J. & WuÈ thrich, K.

(1980) High-resolution 1H-NMR studies of

self-aggregation of melittin in aqueous

solution. Biochim. Biophys. Acta. 622, 231±

244.

24. Hirota, N., Mizuno, K. & Goto, Y. (1997)

Cooperative alpha-helix formation of beta-

lactoglobulin and melittin induced by

hexa¯uoroisopropanol. Protein Sci. 6, 416±

421.

25. Lauterwein, J., Brown, L.R. & WuÈ thrich, K.

(1980) High-resolution 1H-NMR studies of

monomeric melittin in aqueous solution.

Biochim. Biophys. Acta. 622, 219±230.

26. Inagaki, F., Shimada, I., Kawaguchi, K.,

Hirano, M., Terasawa, I., Ikura, T. & Go, N.

(1989) Structure of melittin bound to

perdeuterated dodecylphosphocholine

micelles as studied by two-dimensional NMR

and distance geometry calculations.


27. Wishart, D.S., Sykes, B.D. & Richards, F.M.

(1991) Relationship between nuclear

magnetic resonance chemical shift and

protein secondary structure. J. Mol. Biol. 222,

311±333.

28. Guntert, P., Qian, Y.Q., Otting, G., Moller,

M., Gerhring, W.J. & WuÈ thrich, K. (1991)

Structure determination of the Antp (C39ÐS)

homeodomain from nuclear magnetic

resonance data in solution using a novel

strategy for the structure calculation with the

programs DIANA, CALIBA, HABAS and

GLOMSA. J. Mol. Biol. 217, 531±540.

29. Guntert, P., Mumenthaler, C. & WuÈ thrich, K.

(1996) Dyana: torsion angle dynamics for

structure calculation and automatic

assignment of NOESY spectra: Abstract.

XVIIth International Conference on

Magnetic Resonance in Biological Systems,

Keystone, Colorado, USA.

30. Dill, K.A. (1990) The meaning of

hydrophobicity. Science 250, 297±298.

31. Privalov, P.L. & Gill, S.L. (1988) Stability of

protein structure and hydrophobic

interaction. Adv. Protein Chem. 39, 191±234.

32. Kirk-Othmer (1980) Encyclopedia of

Chemical Technology, Vol. 11, 3rd edn. John

Wiley and Sons, New York, pp. 2±24.

33. Guo, W., Li, Z., Fung, B.M., O'Rear, E.A. &

Harwell, J.H. (1992) Hybrid Surfactants

containing separate hydrocarbon and

¯uorocarbon chains. J. Phys. Chem. 96, 6738±

6742.

34. Muller, N. & Platko, F.E. (1971) Investigation

of micelle structure by ¯uorine magnetic

resonance. IV. Fluorine-labelled non-ionic

detergents. J. Phys. Chem. 75, 547±553.

35. Andersen, N.H., Cort, J.R., Liu, Z., Sjoberg,

S.J. & Tong, H. (1996) Cold denaturation of

monomeric peptide helices. J. Am. Chem.

Soc. 118, 10309±10310.

36. Georghiou, S., Thompson, M. &

Mukhopadhyay, A.K. (1982) Melittin±

phospholipid interaction studied by

employing the single tryptophan residue as

an intrinsic ¯uorescent probe. Biochim.

Biophys. Acta 688, 441±452.

37. Cruz, A., Casals, C. & Perez-Gil, J. (1995)

Conformational ¯exibility of pulmonary

surfactant proteins SP-B and SP-C, studied in

aqueous organic solvents. Biochim. Biophys.

Acta 1225, 68±76.

38. Chen, Y., Liu, B. & Barkley, M.D. (1995)

Tri¯uoroethanol quenches indole

¯uorescence by excited-state proton transfer.

J. Am. Chem. Soc. 117, 5608±5609.

39. WuÈ thrich, K. (1986) NMR of Proteins and

Nucleic Acids. Wiley, New York.

40. Pardi, A., Wagner, G. & WuÈ thrich, K. (1983)

Protein conformation and proton nuclear-

magnetic-resonance chemical shifts. Eur. J.

Biochem. 137, 445±454.

41. Dempsey, C.E. (1988) pH Dependence of

hydrogen exchange from backbone peptide

amides of melittin in methanol.


42. Dempsey, C.E. (1992) Quantitation of the

effects of an internal proline residue on

individual hydrogen bond stabilities in an

alpha-helix: pH-dependent amide exchange in

melittin and [Ala-14]melittin. Biochemistry

31, 4705±4712.

43. MacArthur, M.W. & Thornton, J.M. (1991)

In¯uence of proline residues on protein

conformation. J. Mol. Biol. 218, 397±412.

44. Ramachandran, G.N., Ramakrishnan, C. &

Sasisekharan, V. (1963) Stereochemistry of

polypeptide chain con®gurations. J. Mol. Biol.

7, 95±99.

45. Hurley, J.H., Manson, D.A. & Matthews,

B.W. (1992) Flexible-geometry

conformational energy maps for the amino

acid residue preceding a proline. Biopolymers

32, 1443±1446.

46. Brown, L.R., Braun, W., Kumar, A. &

WuÈ thrich, K. (1982) High resolution nuclear

magnetic resonance studies of the

conformation and orientation of melittin

bound to a lipid±water interface. Biophys. J.

37, 319±328.

47. Tatham, A.S., Hider, R.C. & Drake, A.F.

(1983) The effect of counterions on melittin

aggregation. Biochem. J. 211, 683±686.

48. Juvvadi, P., Vunnam, S. & Merri®eld, R.B.

(1996) Synthetic melittin, its enantio, retro,

and retroenantio isomers, and selected

chimeric analogs: their antibacterial,

hemolytic, and lipid bilayer action. J. Am.

Chem. Soc. 118, 8989±8997.

49. Kentsis, A. & Sosnick, T.R. (1998)

Tri¯uoroethanol promotes helix formation

by destabilizing backbone exposure:

desolvation rather than native hydrogen

bonding de®nes the kinetic pathway of

dimeric coiled coil folding. Biochemistry 37,

14613±14622.

50. Hirota, N., Mizuno, K. & Goto, Y. (1998)

Group additive contributions to the alcohol-

induced alpha-helix formation of melittin:

implication for the mechanism of the alcohol

effects on proteins. J. Mol. Biol. 275, 365±

37851.


J. Peptide Res. 54, 1999 / 100±111 | 111

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