Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology

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Effects of Fluorination on the Bioorganic Properties of Methionine 461

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49.

Structure Analysis of Membrane - Active

Peptides Using

F - labeled Amino

Acids and Solid - State NMR

Parvesh Wadhwani and Erik Strandberg

18.1 Introduction

Fluorine, one of the most abundant elements on earth, is rarely found to participate in

biological processes. With its small size and high electronegativity, the physiochemical

and pharmacological properties of ﬂ uorinated molecules can be widely tuned to obtain

substances that range from metabolically stable drugs to very poisonous compounds.

During the last two decades, there has been an increasing use of ﬂ uorine in bioorganic

and medicinal chemistry [1, 2] . Incorporation of ﬂ uorine or a CF

- group as a hydrogen

atom replacement has led to a substantial increase in the bioactivity and bioavailability

of a variety of pharmaceuticals. Similarly, there has been an increase in the use of ﬂ uorine

in other areas of chemistry, such as agricultural and materials sciences. Likewise, the high

sensitivity of ﬂ uorine as an NMR - active nucleus and the large

F –

H or

F –

F dipolar

coupling strengths have also found tremendous application. Consistent development

of new synthetic methods to produce

F - labeled amino acids, complemented by simultane-

ous development in the ﬁ eld of

F NMR hardware, has led to a situation in which

NMR can be routinely used to study

F - labeled peptides and proteins and address a

variety of open questions [3 – 7] . By performing

F NMR on a suitably labeled peptide or

protein, one can easily obtain information about conformational changes, the structure and

orientation of peptides, or the kinetics of ligand binding, properties that are intimately

related to the function of the peptide. The pre - requirement of such experiments is the

Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima

464 Fluorine in Medicinal Chemistry and Chemical Biology

inclusion of a speciﬁ c

F - reporter group. This chapter highlights the use of

F - labeled

amino acids and their incorporation into peptides and proteins using biosynthetic and

chemical approaches. A description is given of solid - state

F NMR experimental proce-

dures and how experimental data may be used to determine the structure and orientation

of peptides in membranes, giving information that is fundamental to understanding and

improving their function. Finally, some results are presented where several membrane -

active peptides prepared with various

F - labeled amino acids have been investigated using

solid - state

F NMR. The chapter focuses on the use of orientational NMR constraints and

will not go into details about NMR distance constraints.

18.1.1 Fluorine - containing Peptides

Incorporation of ﬂ uorine into the peptides can be straightforwardly achieved using

F -

labeled amino acids and synthesizing the peptide in question by following standard solid -

phase peptide synthesis (SPPS) protocols. There have been numerous reports on the

synthesis of

F - labeled amino acids that are beyond the scope of this chapter and have

been described in detail elsewhere [1, 2] . Generally, a

F - labeled amino acid is an ana-

logue of a naturally occurring amino acid. Fluorine is the element with the second smallest

known atomic radius, and the replacement of a hydrogen atom by a ﬂ uorine atom is

expected to cause minimum steric perturbations; therefore, a variety of amino acids can

be synthesized in which a single hydrogen atom is replaced with a ﬂ uorine atom in the

side chain. Alternatively,

F - labeled peptides may also be obtained by direct ﬂ uorination

of peptides, using a variety of ﬂ uorinating agents, but most of these efforts have been

aimed to ﬂ uorinate cyclic or aromatic amino acids (see reference 8 and references

therein).

For example, synthesis of α - ﬂ uoroglycine has been attempted by various researchers,

but this amino acid is unstable and spontaneous elimination of HF results in an

acyl - iminium intermediate that further decomposes; however, syntheses of protected α -

ﬂ uoroglycines have been reported [9] . Most of the efforts so far have concentrated on the

synthesis of either β - ﬂ uoro - amino acids, such as β - ﬂ uoroalanine and their di - and triﬂ uoro

derivatives, or of γ - ﬂ uorinated amino acids, as discussed in detail in the literature [9, 10] .

Replacement of a methyl group (CH

- , with a volume of 54 Å

) by a triﬂ uoromethyl group

(CF

- , with a volume of 94 Å

) leads to some structural changes due to the increased spatial

requirement, but often such substitutions are justiﬁ ed by comprehensive structural and

functional studies on the peptide in question [11, 12] . Substitution of a CH

- group by a

- group in alanine, isoleucine, leucine and valine is well known. These

F - labeled

analogues have been found to change the solubility properties of peptides or, for example,

to enhance the stability of coiled coil motifs (see references 12 – 16 and references

therein).

Several aromatic amino acids have also been converted to their

F - labeled analogues

by replacement of a single H atom on the aromatic ring. These include ﬂ uorotryptophan,

ﬂ uorophenylalanine, ﬂ uorophenylglycine, ﬂ uorotyrosine, and their various isomers.

Certain solid - state

F NMR structural approaches require speciﬁ c

F - labeled amino acids

bearing a ﬂ uorine atom or CF

- group that is rigidly attached to the peptide backbone

(collinear with the C

– C

bond). These include 4 - F - phenylglycine (4F - Phg), 4 - CF

Structure Analysis of Membrane Active Peptides 465

phenylglycine (CF

- Phg), 3 - CF

- bicyclopentylglycine (CF

- Bpg), and 2 - CF

- alanine ana-

logues. For other analytical methods of investigation (solution NMR, circular dichroism

[CD], analytical centrifugation, and thermodynamic methods) several other

F - labeled

amino acids have been employed [17 – 22] . These

F - labeled amino acids may contain a

single ﬂ uorine atom next to a hydroxyl group (i.e., serine and threonine), a thiol group

(cysteine and methionine), a carboxyl group (glutamic acid and aspartic acid), or a nitrogen

functionality (glutamine, histidine, lysine, proline, and tryptophan). Likewise, there are

reports of triﬂ uoro - and hexaﬂ uoro - derivatives of isoleucine, leucine, and valine having

been employed in protein design and engineering [12 – 16] .

18.1.2 Membrane - Active Peptides

The cellular membrane is a hydrophobic barrier that surrounds the cytoplasm of every cell

and is involved in complex cellular processes, such as signaling and transport, which are

essential to maintain the normal life cycle of a cell; major components of this cellular

membrane are lipids, proteins, peptides, and carbohydrates. Peptides that interact with

cellular membranes are referred to as membrane - active peptides and can be broadly

divided into three major classes: antimicrobial, cell - penetrating, and fusogenic peptides.

Any of these may have variable lengths, hydrophobicities, and secondary structures, but

they often exhibit similar effects on membranes. For example, some antimicrobial peptides

have cell - penetrating properties, and vice versa [23, 24] ; these peptides usually cause some

degree of membrane destabilization.

Antimicrobial peptides (AMPs), or host defense peptides, are short (10 – 40 amino

acids) and positively charged and they usually form an amphipathic structure ( α - helix, β -

sheet, cyclic or ﬂ exible). Upon microbial invasion, these peptides move into action and kill

the invading microbes by rupturing their membrane or by making pores in the membrane

[25 – 27] . In higher organisms, they can also activate further host defense pathways. Several

studies suggest that these peptides are initially unstructured in solution. Upon membrane

binding, which is governed by initial electrostatic attraction between the positively charged

peptide and the negatively charged bacterial membrane, these peptides acquire their sec-

ondary amphipathic structure and lie on the membrane surface in an orientation that com-

pensates for charge and hydrophobicity (see Figure 18.1 ). As the concentration of the

peptides is increased, they either break the membrane barrier ( “ carpet mechanism ” [28] )

or insert into the membrane and form organized peptide assemblies which permeabilize

the membrane by forming pores. In the “ barrel - stave pore ” model, peptides tilt into a

transmembrane orientation and form a bundle, resulting in a hole in the membrane and

causing cytoplasmic leakage [29] . In the “ toroidal wormhole pore ” model, some negatively

charged lipid molecules also participate in the formation of a hole, thereby compensating

the charges of the peptides [30] . Figure 18.1 illustrates some of the models that are often

invoked to explain the mechanism of action of AMPs. The peptides often also show hemo-

lytic activity against human erythrocytes, which has so far limited their therapeutic use.

Cell - penetrating peptides (CPPs) can internalize into cells without disturbing the

membrane potential and, therefore, are an attractive research target for drug delivery. These

peptides are also short, cationic, and usually amphiphilic, and are able to bring a “ func-

tional cargo ” into the cell [24, 31 – 33] . There is no mechanism unanimously agreed upon,

466 Fluorine in Medicinal Chemistry and Chemical Biology

and various cellular processes related to endocytosis may be involved. For direct peptide

uptake across the plasma membrane, or just as well for escape from an endosome, an

inverted micellar mechanism has been proposed to explain the release of cargo into the

cytosol (see Figure 18.1 ).

Fusogenic peptides, on the other hand, are instrumental at the membrane surface and

mediate the fusion of two lipid bilayers [34] , which is vital for fertilization and is an

essential step in various viral infections. Figure 18.2 illustrates how the two opposing

membranes are perturbed by the presence of a fusogenic peptide to form an intermediate

state, which then results in complete intermixing of the cytoplasmic material. In nature

these peptides are usually smaller fragments of larger proteins, but in vitro the smaller

fragments are self - sufﬁ cient in executing membrane fusion.

Folding

Binding

Peptide carpet

Toroid wormhole pore

Inverted micelle

Barrel-stave pore

Micellization

Figure 18.1 Models for different modes of peptide – lipid interaction of membrane - active

peptides. The peptide remains unstructured in solution and acquires an amphipathic structure

in the presence of a membrane. The hydrophobic face of the amphipathic peptide binds to

the membrane, as represented by the grayscale. At low concentration, the peptide lies on the

surface. At higher peptide concentrations the membrane becomes disrupted, either by

the formation of transmembrane pores or by destabilization via the “ carpet mechanism. ” In

the “ barrel - stave pore ” the pore consists of peptides alone, whereas in the “ toroidal wormhole

pore ” negatively charged lipids also line the pore, counteracting the electrostatic repulsion

between the positively charged peptides. The peptide may also act as a detergent and break

up the membrane to form small aggregates. Peptides can also induce inverted micelle structures

in the membrane.

Structure Analysis of Membrane Active Peptides 467

18.1.3 Structure and Orientation of Peptides in Membranes

To understand the function of membrane - active peptides, it is important to know the

structure and orientation of the peptide in the membrane. As is evident from Figure 18.1 ,

it is possible to distinguish between, for example, carpet and pore mechanisms of action

by determining the peptide ’ s orientation in the membrane. Various techniques, such as

electron spin resonance (ESR) [35] , infrared (IR) spectroscopy [36 – 38] , circular dichroism

(CD) [35, 39, 40] , and solid - state NMR (SSNMR) [4 – 7] are used to investigate membrane -

active peptides in a quasi - native lipid bilayer environment. In the following sections,

methods to determine peptide structure and orientation are presented.

18.1.3.1 Peptide Structure

The ﬁ rst step in these investigations is usually to determine or to conﬁ rm the secondary

structure of the peptide. The conventional methods used to determine the three -

dimensional structure of biomolecules are x - ray crystallography and solution - state NMR,

but neither method is suitable for small peptides bound to membranes. While it is relatively

easy to crystallize peptides by themselves, it is very difﬁ cult to crystallize them in a mem-

brane, due to the high intrinsic mobility of the system. It is easier and more meaningful

to crystallize large membrane proteins, which are less mobile and whose structure is more

determined by internal constraints. Solution - state NMR works well with peptides in solu-

tion or in small detergent micelles that tumble sufﬁ ciently rapidly, but this conformation

can be very different from the structure in a membrane and no orientational information

is gained. SSNMR does not have these limitations and is one of the best methods for study

of the structure of peptides in membranes [41 – 43] . SSNMR usually requires isotopic

peptide labeling, and, in order to determine a detailed structure, a large number of con-

straints are needed.

Mixing

(a) (b)

(c)

Figure 18.2 (a) Two membranes and a fusogenic peptide before fusion. (b) Fusion of the

two lipid bilayers, mediated by fusogenic peptides. (c) Merged compartments after fusion.

468 Fluorine in Medicinal Chemistry and Chemical Biology

The structure of a peptide inside a hydrophobic membrane is highly constrained.

There is a high cost of free energy for exposing polar groups of the peptide backbone,

such as CO and NH, to the hydrophobic environment; therefore, these groups need to form

hydrogen bonds. In the membrane, such hydrogen bonds are most readily accommodated

in regular secondary structures, namely α - helices or β - sheets. In fact, most transmembrane

domains of proteins with known structure are α - helical, and there are also a few integral

membrane proteins forming β - sheet structures, for example the β - barrel porins. As a ﬁ rst

approximation of the peptide structure, an α - helical or a β - stranded type of structure can

readily be identiﬁ ed by CD in lipid vesicles. It is also possible to use solution - state NMR

methods to determine the peptide structure in micelles to serve as a model for the structure

analysis in lipid membranes. For some peptides with strong internal constraints, such as

small cyclic peptides or peptides with one or more disulﬁ de bridges, it is most meaningful

to obtain the structure from solution - state NMR or x - ray crystallography, and it is usually

safe to assume that the resultant highly stable structure will be the same as in the

membrane.

18.1.3.2 Peptide Orientation

Once the structure of a membrane - active peptide is known, it is then possible to determine

the orientation of the peptide in the membrane. This orientation can be deﬁ ned in terms

of a tilt angle ( τ ) and an azimuthal angle ( ρ ) (see Figure 18.3 ). The tilt angle is the angle

between the peptide ’ s long axis and the bilayer normal; for helical peptides, the helix axis

is conveniently deﬁ ned as the long axis. The azimuthal angle describes the rotation around

the long axis, deﬁ ned from some individually deﬁ ned point of reference. The two most

S-state T-state I-state

Figure 18.3 The orientation of a peptide in the membrane can be described by the tilt angle

τ and the azimuthal angle ρ . τ is the angle between the bilayer normal ( n ) and the peptide

long axis. ρ describes a rotation around the peptide long axis and must be deﬁ ned with respect

to a reference group as indicated by the white circle. In liquid - crystalline bilayers, peptides

can usually also rotate around the membrane normal (shown by the dashed arrow). Three

characteristic peptide orientations are shown: in the S - state the peptide lies ﬂ at on the

membrane surface with charged amino acids facing the water; in the T - state the peptide is

inserted with an oblique tilt into the membrane, possibly in a dimeric state (shown as a second

peptide in white); and in the inserted I - state the peptide has a transmembrane orientation. In

this state, the peptide may self - assemble into pores (shown here as a barrel - stave pore together

with additional white peptides).

Structure Analysis of Membrane Active Peptides 469

extreme orientations of membrane - active peptides distinguish between peptides lying ﬂ at

on the membrane surface, and inserted peptides with a transmembrane orientation. The

ﬁ rst case is referred to as the “ surface ” state or S - state, and the second case is called the

“ inserted ” state or I - state. In the S - state, the tilt angle is approximately 90 ° and in the I -

state it is approximately 0 ° . From SSNMR on membrane - active peptides, an obliquely

“ tilted ” state has also been detected in several cases; this is called the T - state, in which

peptides are tilted by approximately 125 ° [44 – 47] . These three states are shown in Figure

18.3 .

Several methods are available for measuring the orientation of a membrane - active

peptide in a lipid bilayer and each of these differs in accuracy. One such method is oriented

circular dichroism (OCD), which gives an approximate tilt angle of α - helical peptides,

and which can monitor the reorientation of β - sheet peptides [39] . This method does not

require any labeling, but on the other hand it provides no information about the azimuthal

angle. More accurate values of the tilt, as well as the azimuthal angle, can be obtained

using SSNMR [4] . Many NMR parameters depend on the orientation of certain NMR

tensors with respect to the static magnetic ﬁ eld, which provides a natural reference frame.

Examples of such tensors are the chemical shift anisotropy, dipolar and quadrupolar

tensors [48] . These tensors are ﬁ xed in the peptide molecule and, by determining the ori-

entation of several tensors with respect to the magnetic ﬁ eld, it is possible to calculate the

orientation of the molecules with respect to the magnetic ﬁ eld and also to the membrane.

This process is described in more detail in the next section. In this chapter we will focus

on the use of solid - state

F NMR to characterize

F - labeled peptides in model

membranes.

18.2 Solid - State

F NMR of Membrane - Active Peptides

This section brieﬂ y describes the

F NMR experiments performed on

F - labeled peptides,

the fundamental NMR parameters that are used to obtain orientational constraints, and the

means by which peptide structure is determined from these constraints.

18.2.1 Advantages of

F NMR

SSNMR methods usually rely on isotopic labeling of the peptide, in contrast to solution -

state NMR which often makes use of naturally occurring

H nuclei. The dipolar couplings

between these protons are motionally averaged in solution, but this is not the case in the

solid state. These strong interactions give broad lines in SSNMR spectra, which makes it

impossible to resolve the signals of individual

H nuclei. Accordingly, other NMR - active

nuclei are introduced into the molecule and observed by SSNMR. The most common iso-

topes used for labeling are

C and

H. However, the ﬁ rst has low sensitivity, and the

other two have considerable natural abundance, which can give problems with background

signals, especially at low concentrations of the peptide of interest. Instead, for studies of

membrane - active peptides our group has developed methods using

F as an NMR label

(see references 6, 7 and references therein) with high sensitivity and no background

470 Fluorine in Medicinal Chemistry and Chemical Biology

signals. Table 18.1 summarizes advantages and disadvantages of some selected NMR

labels. It is evident from Table 18.1 that using highly sensitive

F - labels it is possible to

investigate concentration regimes that are not easily accessible using conventional isotope

labels. It is also possible to use smaller amounts of material or shorter measurement times.

F NMR offers complete information about peptide orientation and dynamics.

Hardware requirements are slightly different from those for traditional NMR mea-

surements; NMR probe heads must be absolutely free from ﬂ uorine, but such probes are

now commercially available. It is usually necessary to have two high - frequency channels

on the NMR spectrometer to allow ﬂ uorine measurements with proton decoupling, and

good RF - ﬁ lters must be used to separate the

F and

H channels.

18.2.2 Solid - State NMR Constraints

In order to determine the structure and alignment of a peptide, it is necessary to determine

the orientation of several NMR interaction tensors in the magnetic ﬁ eld; however, ﬁ rst the

orientation of the tensors should be known in the molecular frame. The peptide structure

and its alignment in the membrane can then be determined, given that the membrane ori-

entation in the magnetic ﬁ eld is known. Thus, it is often advantageous to use macroscopi-

cally oriented membrane samples in which the lipid membrane normal is aligned parallel

to the external magnetic ﬁ eld.

In the case of

F - labels, the relevant tensors are the

F chemical shift anisotropy

(CSA), and for CF

- groups the

F –

F homonuclear dipolar couplings. Both interactions

have a ½(3 cos

θ − 1) dependence, where θ is the angle between the interaction tensor and

the magnetic ﬁ eld direction. Both tensors can be used to determine peptide orientation;

however, the orientation of the CSA tensor is not well characterized in all

F - labeled

amino acids (see e.g. 4F - Phg). In practice, it is also difﬁ cult to measure the chemical shift

accurately, since referencing of

F NMR signals is not straightforward [49] ; it is therefore

often easier to use the dipolar couplings of CF

- groups. The orientation of the dipolar

Table 18.1 Comparison of selected NMR labels used for peptide structure analysis

Information Nucleus

Label

N in backbone Ala - d

- group

Sensitivity

Low ( < 1%)

Medium (5%) High (100%)

Minimum * P/L

1 : 200 1 : 300 1 : 3000

Structure analysis Single label gives helix

tilt , but azimuthal

rotation is not

generally accessible

Ambiguous sign of

splitting can give

multiple solutions,

hence many

labels are required

Full information on

tilt and azimuthal

rotation, but several

labels are required

Accuracy

Low ( ± 30 ° )

High ( ± 5 ° )

Medium ( ± 15 ° )

Structural

perturbation

Entirely unperturbing Unperturbing at

alanine positions ,

risky at other

positions

Risk of perturbation

* P/L

= peptide-to-lipid molar ratio