Improved Solid-Phase Peptide Synthesis of Wild-Type and Phosphorylated Phospholamban Using a Pseudoproline Dipeptide

doi:10.4236/ojsta.2012.12002

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Open Journal of Synthesis Theory and Applications, 2012, 1, 9-12

http://dx.doi.org/10.4236/ojsta.2012.12002 Published Online July 2012 (http://www.SciRP.org/journal/ojsta)

Improved Solid-Phase Peptide Synthesis of Wild-Type

and Phosphorylated Phospholamban Using

a Pseudoproline Dipeptide

Shadi Abu-Baker*, Gary A. Lorigan

Department of Chemistry and Biochemistry, Miami University, Oxford, USA

Email: *[email protected]

Received April 2, 2012; revised May 17, 2012; accepted June 13, 2012

ABSTRACT

In this study, we report that the insertion of a pseu doproline dipeptide for the solid-phase peptide synthesis of wild-type

Phospholamban protein (WT-PLB) has two important advantages. First, it disrupts the formation of different secondary

structures, which is responsible for poor couplings during the preparation of highly aggregated sequences. Second, it

enhances the purities and solubility of crude products leading to easier HPLC purification.

Keywords: Solid-State Peptide Synthesis; Pseudoproline Dipeptide; Phospholamban

1. Introduction

Phospholamban (PLB) is a hydrophobic 52-amino acid

transmembrane protein that is involved in regulating the

contraction and relaxation of heart muscle [1-3]. Phos-

phorylation of PLB by cyclic AMP- and calmodulin-

dependent kinases is believed to increase the rate of cal-

cium re-uptake by the sarcoplasmic reticulum and result

in muscle relaxation [1-3]. The isolation and purification

of large quantities of native PLB through molecular bi-

ology techniques has not yet been achieved due to diffi-

culties encountered in the bacterial over expression of

phospholamban cDNA [4,5]. Alternatively, PLB has been

prepared by chemical synthesis using standard solidphase

peptide synthesis and purification in organic solvents [6,

7]. In addition, this approach gives the opportunity to

synthesize site-specific isotopically labeled peptides and

proteins [8-10]. The biochemical and biophysical com-

parison of synthetic PLB and native PLB revealed that

they are both similar in size and functionally identical

[6,7].

2. Materials and Methods

2.1. WT-PLB Synthesis and Purification

PLB was synthesized using modified Fmoc-based solid-

phase methods with an ABI 433A peptide synthesizer

(Applied Biosystems, Foster city, CA). During our first

run we found that the coupling of Leu-7 to Thr-8 was

difficult even after double coupling and extending the

reaction time to six hours. However, this problem was

solved by using the pseudoproline dipeptide of Fmoc-

Leu-Thr (Me,Me Pro)-OH from Novabiochem (San Die-

go, CA). The use of a pseudoproline dipeptide of Fmoc-

Leu-Thr (Me,Me Pro)-OH enhanced the yield to about 25

% after lyophylization. To synthesize P-PLB, a pre-phos-

-phorylated Fmoc-serine amino acid was used at amino

acid position 16 instead of the regular Fmoc-serine used

in the synthesis of PLB. The crude peptide was purified

on an Amersham Pharmacia Biotech AKTA explorer 10S

HPLC controlled by Unicorn (version 3) system software.

The purified protein was lyophilized and characterized by

matrix-assisted laser desorption ionization time of flight

(MALDI-TOF) mass spectrometry.

3. Results

3.1. Solid-Phase Peptide Synthesis of WT-PLB

The chemically synthesized form of the full length PLB

(Figure 1(a)) and P-PLB (Figure 1(b)) was used for all

of the solid-state NMR experiments. In general, solid-

phase peptide synthesis (SPPS) starts with the C-terminal

amino acid attached to a solid support (resin). Amino

acids are then coupled one at a time till the N-terminus is

reached. Each time an amino acid is added, the following

three steps are repeated: First, deprotection of the N-

terminal amino acid of the peptide bound to the resin

(removal of the Fomc protecting group, see the aromatic

part in Figure 2). This step is followed by activation and

coupling of the next amino acid. And finally, the new

N-terminal amino acid is deprotected [11].

*Corresponding author.

S. ABU-BAKER, G. A. LORIGAN

H-Met1-Asp-Lys-Val-Gln-Tyr- Arg-Ser10-Ala-Ile-Arg-Ar

g-Ala-Ser-Thr-Ile-Glu-Met20-Pro-Gln-Gln-Ala-Arg-Gln-Asn-Leu-

Gln-Asn30-Leu-Phe-Ile-Asn-Phe-Cys-Leu-Ile-Leu-Ile40-Cys-Leu-

Leu-Leu-Ile-Cys-Ile-Ile-Val-Met50-Leu-Leu-OH

(a)

H-Met1-Asp-Lys-Val-Gln-Tyr- Arg-Ser10-Ala-Ile-Arg-Ar

g-Ala-Ser(PO

)-Thr-Ile-Glu-Met20-Pro-Gln-Gln-Ala-Arg-Gln-

Asn-Leu-Gln-Asn30-Leu-Phe-Ile-Asn-Phe-Cys-Leu-Ile-Leu-Ile40-

Cys-Leu-Leu-Leu-Ile-Cys-Ile-Ile-Val-Met50-Leu-Leu-OH

(b)

Leu-Thr-

Figure 1. Pr imary sequence of (a) PLB and (b) P-PLB. Site s

of pseudoproline substitution are highlighted in red. P-Ser

residue highlighted in blue was introduced using Fmoc-Ser

(PO(OBzl)OH)-OH.

COOH

CH3

Figure 2. The pseudoproline dipeptide F moc -Leu-Thr(CM e,

Mepro)-OH. This structure was generated using Chem-

Draw software and it is similar to the structure shown in

the Novabiochem website [13].

To control the progress of the synthesis, the deprotec-

tion and coupling steps can be monitored using a UV

detector. Several approaches including switching to dif-

ferent resins and activating reagents as well as using a

pseudoproline dipeptide has been suggested to improve

the yield of poor synthesis [12]. Figure 2 shows the

pseudoproline dipeptide Fmoc-Leu-Thr(CMe,Mepro)-OH.

In this dipeptide, the Thr residue has been reversibly

protected as proline-like TFA-labile oxazolidine [13 ].

WT-PLB was synthesized according to a new proce-

dure developed in the Lorigan’s lab. Briefly, WT-PLB

was synthesized using modified Fmoc-based solid-phase

methods with an ABI 433A peptide synthesizer (Applied

Biosystems, Foster city, CA). WT-PLB is very hydro-

phobic; thus, the synthesis of this peptide is very chal-

lenging. Nevertheless, by using a combination of ex-

tended coupling and deprotection protocols with a single

pseudoproline dipeptide substitution, we were able to

obtain both purified PLB and P-PLB in a yield of 25%.

Couplings were performed using 10-fold excess of Fmoc-

amino acids activated with HBTU/DIPEA. The synthe-

sizer was programmed to use conditional UV feedback

monitoring; coupling and deprotection reactions are ex-

tended automatically, and a capping step introduced after

the coupling step, based on the kinetic profile of the

Fmoc deprotection reaction. For certain residues addi-

tional extensions to the coupling times were used as in-

dicated in Table 1.

All peptides were cleaved from the resin by treatment

with TFA/EDT/thioanisole/water (10:0.5:0.25:0.5) for

2.5 h, and isolated by centrifugation followed by precipi-

tation with methyl t-butyl ether. PLB consists of a hy-

drophilic N-terminus (residues 1 - 20), a hinge region (21

- 30) and a hydrophobic



-helical transmembrane tail (31

- 52) [1]. From previous work by Lorigan and co-work-

ers [11], it is known that the synthesis of the C-terminal

transmembrane region of PLB is extremely difficult, par-

ticularly the region from Cys36 to Cys45. To overcome

these difficulties, the Lorigan gro up developed a strategy

involving extended double coupling together with cap-

ping and conditional repetition of the Fmoc deprotection

reaction [11]. Using this approach, PLB (24 - 52) seg-

ment was obtained in a purified yield of 37% [11].

Initially, we attempted the synthesis of full length PLB

with standard amino-acid building blocks using the pro-

tocols previously described [11]. A PEG-PS resin (0.22

mmol/g) was selected as the solid support to reduce steric

crowding and aggregation during chain assembly. Using

the conditional feedback monitoring, this synthesis was

completed in 9 days, as compared to 10 days for the

shorter PLB ( 2 4 - 52) prepare d o n p ol ystyrene resi n [ 1 1].

UV monitoring of the Fmoc deprotection reactions in-

dicated that the peptide assembly proceeded smoothly

until Leu-7 (Figure 3(a)). However, following introduc-

tion of this residue, there was a marked decrease in the

height of the Fmoc deprotection peak, indicating difficul-

ties in the coupling of Leu-7 to Thr-8. Attempts to im-

prove this coupling by double coupling or extending the

reaction time to 6 hours had little effect. In view of the

problems with the coupling of Leu-7 to Thr-8, the syn-

thesis was repeated in exactly the same manner, except

that Leu-7 and Thr-8 were introduced simultaneously

using the pseudoproline dipeptide Fmoc-Leu-Thr(CMe,

Mepro)-OH (Figure 2). In the presence of this dipeptide,

UV monitoring of the Fmoc deprotection reactions indi-

cated that the peptide assembly proceeded reasonably

smoothly until the end of t his synthesis (see Figure 3(b)).

Table 1. Coupling protocols used for assembly of PLB pep-

tides.

Cycle Method

2 - 5 Single coupling

6, 20 - 26, 28, 29 Single coupling + 1 h extension

7 - 14 Double coupling + 6 h ex tension

15 - 19, 31, 34 - 39,

41, 42, 47, 49 - 51 Double coupling

27, 30, 33, 40,

43 - 46, 48 Double coupling + 2 h extension

S. ABU-BAKER, G. A. LORIGAN 11

(a)

(b)

Figure 3. Traces from the UV monitoring of Fmoc removal

during the synthesis of PLB using: (a) Standar d amino acid

building blocks; and (b) A pseudoproline dipeptide (Leu7-

Thr8).

3.2. HPLC Purification of WT-PLB

Following global deprotection and cleavage of the pep-

tide from the resin, PLB was purified by preparative re-

verse phase chromatography (Figure 4) on a C4 column

eluted with a gradient formed between 0.1% TFA in

nanopure water (solvent A) and MeCN/isopropyl alco-

hol/water/TFA (38:57:5:0.1) (solvent B). After lyophili-

zation and using standard Fmoc-amino acid building

blocks (see Figure 4(A)), the purified peptide was ob-

tained in a yield of only 9% based on initial resin substi-

tution. Conversely, with the dip eptide (Figure 4 (B)), the

purified PLB was obtained in a yield of 25%, nearly a

3-fold increase when compared to the synthesis using

standard building blocks.

3.3. Characterization of WT-PLB Using

MALDI-TOF

When the dipeptide was used to synthesize WT-PLB, a

correct mass of 6080 MU was obtained after the purifica-

tion step (Figure 5(A)). Conversely, when the dipeptide

was not used, MALDI-TOF indicated the presence of an

impurity with a mass of 5144 MU, which could be as-

cribed to Ac-PLB (9-52) (Figure 5(B)).

4. Conclusion

The insertion of a pseudoproline dipeptide improved the

synthesis yield and purificatio n of WT-PLB protein. This

insertion has two important advantages. First, it disrupts

the formation of different secondary structures, which is

11001000900800700600500400

(A) Without Dipeptide (PLB)

(B) With Dipeptide (PLB)

Figure 4. Preparative HPLC profiles of (A) WT-PLB pre-

pared using standard Fmoc-amino acid building blocks; (B)

WT-PLB prepared using a pseudoproline dipe ptide; and (C)

The phosphorylated form PLB (P-PLB, a pre-phosphory-

lated Fmoc-serine amino acid was used at amino acid posi-

tion 16 instead of the regular Fmoc-serine used in the syn-

thesis of PLB) prepared using pseudoproline dipeptide.

HPLC conditions: C4 semi-preparative polymer-based col-

umn (259VHP82215, 8 mm 300 Å, 22 mm × 150 mm);

buffer A, 0.1% TFA in water; buffer B, MeCN/isopropyl

alcohol/water/TFA 38:57:5:0.1; gradient, 5% B to 60% B in

25 min then 60% to 100% in 60 min; flow rate, 10 ml/min.

60005000400030002000

(m/z)

(A) WT-PLB (with dipeptide

)

(B) WT-PLB (without dipeptide)

Figure 5. MALDI-TOF spectra of (A) WT-PLB prepared

using a pseudoproline dipeptide; (B) WT-PLB prepared

using standard Fmoc-amino acid building blocks.

S. ABU-BAKER, G. A. LORIGAN

responsible for poor couplings during the preparation of

highly aggregated sequences. Second, it enhances the

purities and solubility of crude products leadin g to easier

HPLC purification. This technique can be used for simi-

lar proteins that show poor synthesis.

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