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

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Structure Analysis of Membrane Active Peptides 471

tensor is well known and it is uniaxial, making calculations easier. The dipolar coupling

can be accurately determined from the splitting in the NMR spectrum, without need for

referencing. In our recent peptide studies we have therefore used various CF

- labeled

amino acids, which will be described below in Section 18.3.2 .

The CF

- group of these amino acids is rigidly attached to the peptide backbone and

reﬂ ects the overall peptide orientation. Due to fast rotation of the CF

- group, the three

pairwise dipolar interactions among the three ﬂ uorine nuclei are all averaged in the same

way and give rise to an average dipolar tensor, which is oriented along the C

– C

- bond

vector. From the measured dipolar coupling, the orientation of the tensor can be deter-

mined, which ﬁ xes the orientation of the bond in the magnetic ﬁ eld [49] . Normally, only

the absolute value of the dipolar coupling can be determined from the spectrum; however,

for a CF

- group the chemical shift change with orientation is of a magnitude similar to

that of the change in dipolar coupling. The sign of the splitting can thus be determined

directly from the position of the chemical shift [49] , as described in Figure 18.4 .

18.2.3 Structure and Orientation Calculations

Using a number of orientational constraints from NMR measurements, the orientation of

the peptide in the membrane can be determined, assuming that the structure of the peptide

is known. The peptides we have studied mostly have well deﬁ ned structures, such as α -

helices or rigid cyclic conformations. The validity of the proposed peptide structure as it

interacts with the membrane can also be tested using the ﬁ t of the data [45, 49] . For

example, the data can be ﬁ tted to different helical models, such as α - and 3

- helix: a good

50 10

0° Tilt

90° Tilt

+6.7 kHz

–3.3 kHz

–30 –70

ppm

–110 –150 50 10 –30 –70 –110 –150

Figure 18.4

F NMR spectra of MSI - 103, where Ile - 13 is replaced with CF

- Phg in DMPC

at P/L = 1 : 400. The left spectrum was measured with the bilayer normal parallel to the

magnetic ﬁ eld and the right spectrum was measured with the bilayer normal perpendicular

to the magnetic ﬁ eld. Because of fast rotation of the peptide around the bilayer normal, the

splittings at are scaled by a factor of − 1/2. The sign of the dipolar coupling can be deduced

from the position of the triplet relative to the isotropic chemical shift (close to − 60 ppm,

marked by an arrow). When the triplet is downﬁ eld of the isotropic chemical shift, the

coupling is positive, and if it is shifted upﬁ eld, the coupling is negative.

472 Fluorine in Medicinal Chemistry and Chemical Biology

ﬁ t can only occur for one structure and will thus give an indication that this is the actual

structure inside the membrane.

In principle, the peptide orientation is fully deﬁ ned by the tilt and azimuthal angles

described in Figure 18.3 . However, peptides interacting with a membrane are mobile, and

to account for this motion a simpliﬁ ed order parameter, S

mol

, is introduced, which has the

effect of scaling all the calculated splittings by a factor between 0 and 1 [4, 23, 44 – 47,

49 – 51] . S

mol

= 0 would correspond to complete isotropic averaging, where all the orienta-

tional information would be lost, and S

mol

= 1 corresponds to a completely immobile

peptide. The peptides in our studies usually have S

mol

values between 0.6 and 0.8. This

gives information about the mobility of the peptides and can also be used to estimate the

size of aggregates. More elaborate motional models have recently been investigated (E.

Strandberg et al. , submitted), but have shown that the calculated values of tilt and azi-

muthal angles for the systems investigated were virtually identical to those obtained using

the more simple approach described here. For our peptides, we thus determine the tilt

angle, the azimuthal angle, and S

mol

. In principle, three constraints are needed to determine

these three parameters, each constraint being obtained from a

F NMR measurement on

a singly

F - labeled peptide. In practice, at least four

F - labeled peptides are used in order

to get a more reliable result, to make sure all the data are consistent, and to rule out any

possible structural perturbations due to introduction of

F - labeled amino acids.

After measuring several local dipolar splittings, these data are used to calculate the global

orientation of the full peptide. From the known structure of the peptide, theoretical curves show

which dipolar couplings are expected for different labeled positions, depending on tilt angle τ ,

azimuthal angle ρ , and S

mol

. The calculated values of splittings are then compared with the

experimentally obtained values, and the root mean square deviation (RMSD) is calculated.

An example of how the procedure works is shown in Figure 18.5 . The peptide in this

example is MSI - 103, which has been labeled with

F at the four positions indicated in

Figure 18.5 a. Four different peptides were synthesized where a single amino acid (Ala - 7,

Ile - 9, Ala - 10, or Ile - 13) was replaced with CF

- Phg. For each label a

F NMR spectrum

is recorded (see Figure 18.5 a); in this example, spectra are from MSI - 103 in DMPC (1,2 -

dimyristoyl - sn - glycero - 3 - phosphocholine) at a peptide - to - lipid molar ratio (P/L) = 1 : 400,

Figure 18.5 (a) The peptide MSI - 103 was labeled with CF

- Phg at four positions, marked in

lighter gray. For each label a

F NMR spectrum was recorded in DMPC at P/L = 1 : 400. From

these spectra the dipolar couplings were measured, giving the values shown next to each

spectrum. (b) The measured dipolar couplings are compared with theoretical curves for

different orientations of the peptide. The best - ﬁ t curve is here shown together with experimental

data for the different labeled positions (ﬁ lled squares). (c) The RMSD plot shows the root

mean square deviation between experimental and calculated splittings, for all possible

combinations of tilt and azimuthal angles. In this case, the best - ﬁ t tilt angle ( τ ) is 101 ° and

the azimuthal angle ( ρ ) is 130 ° . (d) Side view of the peptide in the membrane, which is

represented by a gray box. The tilt angle deﬁ nes the angle between the peptide long axis and

the membrane normal. (e) View of the peptide along the helical axis. The azimuthal angle

deﬁ nes how much the peptide is rotated around its axis, with the starting point deﬁ ned as

the vector from the helical axis to C

of residue Lys - 12 being parallel with the bilayer

surface.

Structure Analysis of Membrane Active Peptides 473

(a)

+1.5 kHz

+6.7 kHz

–2.7 kHz

+2.2 kHz

Ile-9

Ile-13

(b)

(d) (e)

(c)

160

120

–5

–10

–15

Ile-9

Ala-7

Ala-10

RMSD / kHz

0.0 – 1.0

1.0 – 2.0

2.0 – 3.0

3.0 – 4.0

4.0 – 5.0

5.0

50 10 –30 –70

ppm

–110 –150

50 10 –30 –70

ppm

–110 –150

50 10 –30 –70

0 100 200

Position around helix / degrees

Tilt angle / degrees

Dipolar coupling / kHz

300 0 40

τ = 101°

ρ = 130°

Rotation angle / degrees

160120

ppm

–110 –150

50 10 –30 –70

ppm

–110 –150

474 Fluorine in Medicinal Chemistry and Chemical Biology

and dipolar couplings are measured. Each coupling gives information about the orientation

of the respective C

– C

- bond vector of the labeled residue with respect to the magnetic

ﬁ eld. The measured couplings are then used to determine the best - ﬁ t orientation of the

whole peptide. The experimentally observed splittings (ﬁ lled squares in Figure 18.5 b) are

then compared with theoretical splittings, calculated for different peptides orientations.

The x - axis in Figure 18.5 b shows the angle of the label as in a helical wheel projection,

with the ﬁ rst amino acid at 0 ° , the second amino acid at 100 ° and so on; angles larger

than 360 ° are projected back into the 0 – 360 ° region. A change in azimuthal angle will

shift the dipolar curve along the x - axis of the plots, keeping the same shape, while a change

in tilt angle will change the amplitude and shape of the curve. Figure 18.5 b shows the

best - ﬁ t curve of calculated splittings, which corresponds to the darkest area in the RMSD

plot in Figure 18.5 c. In this plot, for each possible combination of τ and ρ , the RMSD

between experimental and calculated splittings is shown in a gray - scale code. In this case,

the best - ﬁ t tilt angle ( τ ) is 101 ° , the azimuthal angle ( ρ ) is 130 ° , and the corresponding

orientation of the peptide in the membrane is illustrated in Figures 18.5 d and 18.5 e.

For a given secondary structure of the peptide, the shape of the dipolar curve is

characteristic of the peptide tilt. Figure 18.6 shows the theoretical dipolar curves for a

surface - bound S - state ( τ ≈ 90 ° ), an inserted I - state ( τ ≈ 0 ° ), and a tilted T - state

(0 ° ≤ τ ≤ 90 ° ) orientation of a helical peptide. The positions in the τ / ρ - plot corresponding

to the different states are indicated. (For exact values of τ and ρ used in the calculations,

see the ﬁ gure legend.) It can be noted that the curve in Figure 18.6 a is very similar to the

curve of Figure 18.5 b, both of which correspond to an S - state orientation of the peptide.

Additional dynamic information can be obtained from oriented samples. The lipid

membranes can be oriented with the bilayer normal either parallel to the magnetic ﬁ eld

(0 ° tilt) or perpendicular to the magnetic ﬁ eld (90 ° tilt). If the peptides rotate quickly

around the bilayer normal, then, according to simple theory of motional averaging, the

measured splittings at 90 ° tilt should be − 1/2 times the splitting at 0 ° tilt. Figure 18.4

shows such

F NMR - spectra of a peptide with fast rotation. On the other hand, if the

peptide does not rotate, then the splitting at 0 ° tilt will be the same, but at 90 ° tilt a

superposition of different orientations around the membrane normal will lead to a more

complex broad lineshape looking like a powder pattern.

If the peptide is not oriented in the membrane but forms large immobilized aggre-

gates, this is easily seen in the NMR spectrum. In this case, all different orientations of

the peptide are present in the sample and a very broad lineshape is seen, as for a peptide

powder. When this is the case, no orientation of the peptide can be determined. Looking

at the NMR spectral lineshape is a useful method for investigating the aggregation behav-

ior of peptides interacting with membranes, which may be intimately related to the func-

tion or malfunction of the peptide.

18.2.4

F NMR Experimental Considerations

To obtain orientational constraints, peptide/lipid samples for

F NMR studies are usually

prepared with lipid bilayers that are macroscopically oriented on glass plates [52 – 54] .

Lipids and peptides in appropriate amounts are co - dissolved in organic solvent and spread

onto thin glass plates. After removal of the solvent, the plates are stacked and placed in a

Structure Analysis of Membrane Active Peptides 475

(a)

–5

–10

–15

0 100 200

Position around helix / degrees

Dipolar coupling / kHz

300

180

140

160

120

100

Tilt angle / degrees

(b)

08040 120

Rotation angle / degrees

160

(c)

–5

–10

–15

0 100 200

Position around helix / degrees

Dipolar coupling / kHz

300

180

140

160

120

100

Tilt angle / degrees

(d)

08040 120

Rotation angle / degrees

160

(e)

–5

–10

–15

0 100 200

Position around helix / degrees

Dipolar coupling / kHz

300

180

140

160

120

100

Tilt angle / degrees

(f)

08040 120

Rotation angle / degrees

160

Figure 18.6 Dipolar wave curves calculated for different orientational states of α - helical

peptides (a, c, e), showing the theoretical dipolar splittings for different positions around the

helical axis. (b, d, f) The orientation of the peptide is determined from a plot of the tilt angle

τ versus the azimuthal angle ρ , where each point in the τ / ρ - plot corresponds to a speciﬁ c

orientation. (a) Helical curve corresponding to an S - state orientation with τ = 90 ° , ρ = 90 ° ;

(b) the position of this S - state orientation in the τ / ρ - plot is marked by the center of the

concentric circles. (c) Helical curve for a tilted T - state orientation with τ = 125 ° , ρ = 90 ° ; (d)

the position of this T - state orientation in the τ / ρ - plot. (e) Helical curve for an I - state orientation

with τ = 10 ° , ρ = 90 ° ; (f) the position of this I - state orientation in the τ / ρ - plot.

476 Fluorine in Medicinal Chemistry and Chemical Biology

hydration chamber to take up water and equilibrate. The hydrated plates are then stacked,

wrapped and stored at − 20 ° C until use. The quality of the peptide/lipid sample is checked

with

P NMR, which gives a signal from the phospholipid head groups from which the

degree of orientation can be estimated, as illustrated in Figure 18.7 .

P NMR also gives

information about the lipid phase behavior and can be used to conﬁ rm that the sample is

in a membrane - like lamellar phase.

F NMR measurements are then conducted with a

H - decoupled single - pulse

F NMR experiment in an NMR probe in which the orientation

of the glass plates can be varied. Usually samples are measured with the bilayer normal

oriented parallel to the magnetic ﬁ eld, as shown in Figure 18.4 for the peptide MSI - 103

labeled with CF

- Phg. A second spectrum can then be acquired with a perpendicular

sample alignment in order to detect the occurrence of long - axis rotation around the mem-

brane normal. Samples can also be prepared as unoriented, multilamellar vesicles (MLV)

by co - dissolving peptides and lipids, and hydrating them after removing the solvent.

If peptides are averaged by long - axial rotation about the bilayer normal, the same

orientational information can be obtained from MLV samples as from oriented samples

[46, 55] .

(a)

(b)

25 0

ppm

–25 –50

50 25 0 –25 –50

Figure 18.7

P NMR spectra of MSI - 103 in DMPC. (a) P/L = 1 : 200, nonoriented

multilamellar vesicles sample. The broad signal shows the typical powder spectrum of a liquid

crystalline lamellar lipid phase. (b) P/L = 1 : 20, macroscopically oriented sample. The peak

around 28 ppm shows that the sample is well oriented with the lipid bilayer normal oriented

parallel to the magnetic ﬁ eld.

Structure Analysis of Membrane Active Peptides 477

18.3

F Labeling of Peptides

F - labeled peptides can be used to study ligand binding, unfolding, mobility, aggregation

and orientation by

F NMR in solution or in their native membrane - bound environment,

and they could also be used to study the effect of ﬂ uorination on thermal stability, solubil-

ity, selectivity, and activity of such biological systems. Highly ﬂ uorinated amino acids

have been employed by various research groups to stabilize proteins for different applica-

tions (see reference 56 and references therein). Since this chapter focuses on investigating

membrane - active

F - labeled peptides by SSNMR, we will restrict ourselves to speciﬁ cally

F - labeled amino acids (see Figure 18.8 ), and their incorporation into peptides at a desired

position. We will then discuss the suitability of various labels for measuring peptide ori-

entation and conformation, mobility, and assembly, and intra - and intermolecular distances

between two ﬂ uorine labels in lipid bilayers.

18.3.1 Labeling Strategies

Incorporation of ﬂ uorine into peptides and proteins is usually achieved through biosyn-

thetic routes, chemical synthesis, or a combination of these two methods. Each method

has its own advantages and shortcomings, but each requires a

F - labeled amino acid. The

most commonly used ones are commercially available analogues of aromatic amino acids,

such as tryptophan, phenylalanine, tyrosine, and phenylglycine. Aliphatic

F - labeled

amino acids are not commonly available and usually have to be synthesized. The synthesis

of most ﬂ uorinated amino acids is described in detail in the literature [1, 9, 10] . For struc-

ture analysis of peptides and proteins, it is important that (i) the ﬂ uorine label is rigidly

attached to the peptide backbone, (ii) the label does not alter the structure or function of

the peptide, (iii) the extent of ﬂ uorination is restricted to avoid multiple signals, and (iv)

Figure 18.8

F - labeled amino acids. Top row: 4F - Phe, 5F - Trp, 4F - Phg, CF

- Phg, CF

- Bpg.

Bottom row: 2D - 3F - Ala, 3F - Ala, F

- Ala, CF

- Ala (R - form), CF

- Ala (S - form).

478 Fluorine in Medicinal Chemistry and Chemical Biology

the label is stable and not prone to chemical side - reactions such as HF - elimination. Figure

18.8 shows various commonly used

F - labeled amino acids.

18.3.1.1 Biosynthetic Labeling

Biosynthetic incorporation of ﬂ uorinated amino acids is a convenient way to obtain

F -

labeled peptides or proteins. With the advancement in biotechnological protocols using

various bacterial and yeast machineries, it has been possible to produce proteins of differ-

ent origin quickly and economically. This process requires an auxotrophic strain of bacteria

for the desired

F - amino acid, which is added to the growth medium. Alternatively,

glyphosate can be used to inhibit the metabolic synthesis of all aromatic amino acids in

bacterial culture so that the desired

F - labeled amino acid along with the other aromatic

ones have to be externally supplemented. Using such approaches, it has been possible to

produce desired sequences with ﬂ uorotryptophan [17, 18] , ﬂ uorotyrosines [57] , ﬂ uorophe-

nylalanine [58, 59] , triﬂ uoroisoleucine [60] , and triﬂ uoroleucine [15] .

Biosynthetic incorporation has several limitations: (i) only ﬂ uorinated analogues

of natural amino acids may be used, provided they are nontoxic to the culture; (ii) site -

selective labeling is not possible; (iii) relatively low uptake of

F - labeled amino acids

results in lower yield of the expressed peptide or protein; and (iv) some proteins might

still contain the natural amino acid that is devoid of ﬂ uorine. All these limitations can be

avoided by using chemical peptide synthesis, as described below.

18.3.1.2 Solid - Phase Peptide Synthesis

Using SPPS protocols,

F - labeled amino acids can be readily incorporated into membrane -

active peptides at any desired site. Standard Fmoc SPPS protocols are detailed in the lit-

erature [61] . Chemical synthesis has an intrinsic limitation in peptide chain length,

depending on the sequence; this limits the synthesis of the ﬂ uorine - containing peptides to

a maximum of 60 amino acids. Most membrane - active peptides have typically 10 – 40

amino acids and are well suited for chemical synthesis. Using synthetic

F - labeled pep-

tides, we have investigated many different systems in model membranes by

F NMR;

these are listed in Table 18.2 , in Section 18.4 . In practice, special care has to be taken at

different stages of the synthetic protocol to avoid problems of ﬂ uorine contamination,

elimination, or racemization, all of which are discussed in the following paragraphs.

Fluorine contamination . Triﬂ uoroacetic acid (TFA), which is used to cleave the full -

length peptide from the solid support and remove the side - chain protections in the ﬁ nal

step of peptide synthesis, is often found to be tightly bound to the peptide. Usually up to

a 5 - fold molar excess of TFA is found to be associated with the peptide, and cannot be

removed by repeated lyophilization [62] . Using an ion - exchange column, it is easy to

replace the triﬂ ate ion with another suitable counteranion, but such puriﬁ cation steps are

accompanied by extensive peptide loss. TFA is also universally used as an ion - pairing

agent in HPLC puriﬁ cation of peptides. For hydrophobic peptides, many researchers use

ﬂ uorinated alcohols, such as triﬂ uoroethanol and hexaﬂ uoroisopropanol, to dissolve the

crude peptide or even as a constituent of the mobile phase during HPLC puriﬁ cation. These

ﬂ uorinated solvents often contaminate the “ puriﬁ ed ” peptide with substantial amounts of

undesired ﬂ uorine, which interferes in the

F NMR spectra. For example, TFA is seen as

an intense signal around − 75 ppm, and this signal overlaps with signals of aromatic ﬂ uorine

Structure Analysis of Membrane Active Peptides 479

and CF

- groups. It is thus essential to exclude any traces of ﬂ uorinated solvents and ion -

pairing agents from the NMR sample. We have shown that TFA from the cleavage step of

peptide synthesis can be efﬁ ciently removed by employing HCl as an ion - pairing agent in

HLPC puriﬁ cation protocols [62] ; therefore, in a single step, it is possible to obtain

F -

labeled peptide that is free of any undesired ﬂ uorine background.

Fluorine elimination. The high electronegativity of ﬂ uorine makes it prone to certain

side reactions that result in HF - elimination. These side reactions are very uncommon for

a ﬂ uorine atom attached to an aromatic ring; however, the presence of labile hydrogen in

the vicinity of an aliphatic ﬂ uorine substituent often leads to spontaneous HF - elimination,

which results either in an oleﬁ nic bond or a cyclized structure. Furthermore, elimination

reactions are accelerated in the presence of base which is typically used in amino acid

coupling procedures. 3 - Fluoroalanine (3F - Ala), or 3,3,3 - triﬂ uoroalanine (F

- Ala) in par-

ticular eliminate HF almost invariably when incorporated via the usual coupling protocols

employing basic conditions. It has been possible to minimize the loss of ﬂ uorine by using

2 - deutero - 3 - ﬂ uoroalanine (2D - 3F - Ala) and by coupling at low temperatures using base -

free coupling strategies [63] .

Racemization. The high electronegativity of ﬂ uorine also makes many

F - labeled

amino acids susceptible to base - induced racemization. In the case of aromatic side - chains,

this effect is even more pronounced, due to the electron - withdrawing effect of the ring.

Under base - catalyzed coupling procedures, ﬂ uorophenylglycine undergoes complete race-

mization, leading to epimeric peptides (see Figure 18.9 ). It has nevertheless been possible

to couple this enantiomerically pure amino acid using base - free protocols employing

diisopropylcarbodiimide/hydroxybenzotriazol to avoid racemization [110] . Most

F -

labeled amino acids are in any case commercially available only as racemic mixtures;

hence the use of standard synthetic protocols involving basic conditions is acceptable and

pragmatic. In this case, the resulting epimeric peptides have to be separated on an HPLC

column and isolated in high purity. In our investigations, we often study both epimeric

Table 18.2 Peptide sequences labeled with

F - labeled amino acids

Peptide

Sequence

Label Reference

Antimicrobial peptides

Gramicidin S Cyclo - (PVOLf)

4F - Phg 62, 66,

- Phg 79

PGLa GMASKAGAIAGKIAKVALKAL - NH

4F - Phg 44, 62

- Phg 44, 49,

84, 85

- Bpg 68

MSI - 103 (KIAGKIA)

- NH

3F - Ala 71

- Phg 45, 89

Cell - penetrating peptide

MAP [110] KLALKLALKALKAALKLA - NH

- Phg Submitted

Fusogenic peptides

B18 LGLLLRHLRHHSNLLANI 4F - Phg 50, 62

FP23 AVGIGALFLGFLGAAGSTMGARS - NH

- Phg Submitted

One - letter code. O = ornithine; f = D - isomer of Phe.

480 Fluorine in Medicinal Chemistry and Chemical Biology

peptides separately after identifying the chirality of the

F - labeled amino acid, in order

to obtain additional structural information [44] . To identify which epimer contains the d -

and l - forms of the

F - labeled amino acid, an aliquot of these separated peptides should

be hydrolyzed to their constituent amino acids and derivatized using Marfey ’ s reagent to

identify the d - and l - forms [62] . Alternatively, the racemic mixture may be acetylated to

obtain the N - acetyl amino acids, which can be subsequently enzymatically deacetylated.

Porcine kidney acylase 1 selectively deacetylates the l - amino acid, leaving the d - enantio-

mer unchanged [64, 65] .

Several different

F - labels with a rigid connection to the peptide backbone, which

qualify for

F NMR according to the criteria described above, were used in our investiga-

tions and will now be described in more detail. These amino acids were incorporated into

various membrane - active peptides as NMR labels, thereby providing structural informa-

tion that will be described in Section 18.4 .

18.3.2

F - labeled Amino Acids

Various

F - labeled amino acids have been used for SSNMR structural studies of

membrane - active peptides. Their chemical structures are shown in Figure 18.8 , including

the aromatic 4 - ﬂ uorophenylalanine, 5 - ﬂ uorotryptophan, 4 - ﬂ uorophenylglycine, and 4 -

triﬂ uoromethylphenylglycine. Apart from the cyclic side chain of 3 - triﬂ uoromethylbicy-

250

(a)

(b)

200

150

100

10 11 12 13

Time (min)

Intensity (220 nm)

14 15

500

400

300

200

10 11 12 13

Time (min)

Intensity (220 nm)

14 15

100

Figure 18.9 (a) HPLC chromatogram showing the presence of two epimeric peptides,

resulting from incorporation of a racemic mixture of CF

- Phg, and (b) analytical HPLC

chromatograms (solid and dashed lines) after preparative separation of the epimers.