Laboratory Experiment pubs.acs.org/jchemeduc
Synthesis, Characterization, and Secondary Structure Determination of a Silk-Inspired, Self-Assembling Peptide: A Laboratory Exercise for Organic and Biochemistry Courses Tyler J. Albin, Melany M. Fry, and Amanda R. Murphy* Department of Chemistry, Western Washington University, Bellingham, Washington 98225, United States S Supporting Information *
ABSTRACT: This laboratory experiment gives upper-division organic or biochemistry undergraduate students a comprehensive look at the synthesis, chemical characterization, self-assembly, and secondary structure determination of small, N-acylated peptides inspired by the protein structure of silkworm silk. All experiments can be completed in one 4 h lab period, followed by two 3 h lab periods, but can be easily tailored to a particular course curriculum, time restraints, or instrument availability. Students synthesize a peptide using solid-phase techniques and characterize the product using nuclear magnetic resonance spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-performance liquid chromatography, and mass spectrometry. Unique from other available peptide-based experiments, self-assembly of the peptide is induced in organic solvent, resulting in the formation of a gel. Following solvent evaporation, FTIR spectroscopy is used to evaluate the secondary structure of the peptide before and after assembly. This set of experiments provides students with an opportunity to explore protein structure from the bottom up, starting from the molecular structure of individual amino acids and continuing through the hydrogen bonding interactions that influence protein 3D structure. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Organic Chemistry, Hands-On Learning/Manipulatives, Amino Acids, Bioorganic Chemistry, NMR Spectroscopy, IR Spectroscopy, Proteins/Peptides, Noncovalent Interactions
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he natural ability of peptides to form well-defined hierarchical structures has recently become a popular target for exploitation in a variety of materials science and biomedical applications. Fibrilliar aggregates and hydrogels formed from peptides and peptide conjugates have been successfully used as biomimetic cell culture scaffolds,1 drug delivery vehicles,2 and stimuli-responsive biomaterials.3,4 Peptides have also been used to control the morphology of larger polymers5,6 and direct the assembly of inorganic nanoparticles to form peptide-based wires7 and sensors.8 Given the prevalence of self-assembling peptides in the literature, this experiment was developed to introduce upperlevel undergraduate students to methods used to synthesize and characterize peptides while highlighting a current research area. The peptide synthesized in this experiment (Figure 1) is modeled after the repetitive glycine-alanine-glycine-alanineglycine-serine (GAGAGS) motif found in silk fibroin produced by Bombyx mori silkworms.9 The GAGAGS domains in silk selfassemble into highly crystalline, antiparallel β-sheets (Figure 2),
Figure 2. Silk structure primarily composed of GAGAGS repeat units that self-assemble into an antiparallel β-sheet.
which are responsible for the characteristic strength of silk fibers. It has been previously shown that a short GAGA sequence is sufficient for promoting β-sheet assembly of alkylated peptides and peptide−polymer conjugates. Molecules containing a GAGA repeat have, therefore, been employed as small-molecule organogelators (form gels in organic solvents),10,11 and have been used to assemble organic semiconductors into fibrils.12 Here, an N-acyl tail was added to the GAGA peptide to increase solubility and aid in characterization. While X-ray crystallography and multidimensional nuclear magnetic resonance (NMR) spectroscopy are commonly employed to determine the 3D structure of proteins, these techniques are time-consuming and require a high level of expertise to interpret the data. Here, Fourier transform infrared
Figure 1. Structure of N-hexyl-GAGA peptide synthesized and evaluated. © XXXX American Chemical Society and Division of Chemical Education, Inc.
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dx.doi.org/10.1021/ed5001203 | J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
phase synthesis methods, and peptide characterization techniques. Students then explore the concept of self-assembly and secondary structure formation and relate this to the individual hydrogen bonding interactions between peptides.
(FTIR) spectroscopy, available in virtually every organic chemistry teaching laboratory, is utilized to probe the secondary structure of the peptides before and after assembly. The vibration of the amide CO in the peptide backbone (often referred to as the amide I band) is particularly sensitive to hydrogen bonds and can be used to identify the presence of different types of secondary structures. Through a compilation of spectra of many well-characterized proteins, a consensus has emerged regarding peak assignments corresponding to β-sheets, α-helices, random coils, and turns, etc. (Table 1).13,14 While
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Overview
Students work in pairs to synthesize N-hexyl-GAGA peptide (Figure 1) using Fmoc-based solid-phase peptide synthesis (SPPS) techniques.19,20 The purity of their products is evaluated using thin-layer chromatography (TLC), 1H NMR spectroscopy, attenuated total reflectance (ATR) FTIR spectroscopy, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). Students then form an organogel by inducing self-assembly of the peptides in tetrahydrofuran (THF) solvent. Here, the organogel is a semisolid solution (gel) containing a hydrogen-bonded network of peptides that traps the organic solvent (THF). An organogel is similar to a hydrogel, but an organic solvent is trapped in the peptide matrix rather than water. To investigate the secondary structure of the peptides in the organogel using ATR-FTIR, the solvent is first removed to form a xerogel (gel without solvent), which retains the hydrogen-bonded structure of the organogel.
Table 1. Consensus Amide CO Vibrations of Proteins in Various Conformations As Measured with FTIR Spectroscopy13−15 Secondary Structure
Vibration, cm−1
β Sheet/extended Parallel Antiparallel α-Helix Random coil/disordered Turns
1621−1640 (strong) 1645 (weak) 1690 (weak) 1651−1662 1638−1655 1663−1696
EXPERIMENT SUMMARY
FTIR analysis of proteins with several different structural domains is quite complex due to overlapping peaks, FTIR analysis can be very useful for simple peptides, such as the one investigated here. Lower CO vibration frequencies are associated with stronger hydrogen bonds. Relevant to this experiment, a prominent shift in the CO vibration from ∼1640 to ∼1625 cm−1 is observed upon transition from a disordered state to a β-sheet structure15 due to the strong hydrogen bonds formed between the peptides. Furthermore, parallel and antiparallel β-sheet structures can often be distinguished by a weak secondary band at 1645 or 1690 cm−1, respectively.14 Using this information, students are able to analyze the intermolecular assembly of their peptides using FTIR data alone. This laboratory experiment was developed specifically for an advanced organic chemistry laboratory course for students majoring in the life sciences and aims to integrate biological aspects of organic chemistry into the curriculum. While a single class can carry out the entire experiment, the experiment could be done as an integrated laboratory experience between organic and biochemistry laboratory courses where organic students complete the synthesis and chemical characterization and then the samples are given to biochemistry students for structural characterization. While this experiment is focused on the synthesis and characterization of the N-acylated hexyl-GAGA peptide (Figure 1), suggestions are provided in the Instructor Notes (see the Supporting Information (SI)) to explore inquiry-based approaches to this experiment by varying the acyl end group. The simplicity of the GAGA repeat, coupled with its welldocumented ability to self-assemble, makes this peptide motif a great learning tool for use in undergraduate organic and biochemistry student laboratories. While other peptide-based laboratory exercises have been published in this Journal,16−18 this experiment is unique in that it allows students to explore intermolecular interactions in peptides, which can further be related to protein folding and three-dimensional (3D) structure. The goals of this experiment are for students to learn the basic molecular structure of amino acids, modern reagents and mechanisms for peptide bond formation, solid-
Organization
The experiment is performed over three lab days, the first requiring about 4 h, followed by two 3 h lab periods. During the first lab period, students carry out the solid-phase synthesis procedure and then use the second day to cleave and isolate the peptides. The peptides are lyophilized by the instructor outside of class after the second lab period. The third day is reserved for characterization and structure analysis. HPLC and MS analyses are carried out after the third lab period, and the results are provided to students for interpretation. Preparation
A plastic syringe (10 mL) with a frit (Torviq brand) is used as the reaction vessel. The syringes are preloaded for students with Fmoc-Ala Wang resin (300 mg). Solutions of 20% piperdine in dimethylformamide (DMF), 25% diisopropylethylamine (DIPEA) in DMF, and solvent mixtures for TLC, HPLC, and MS are premixed for students. Other solvents are dispensed via dispensing pumps preset to the desired volume. A solution of the peptide product (synthesized by a previous class) is provided as a TLC spotting reference. All other reagents are used directly from the manufacturers’ bottles. Procedure Overview
The peptide is synthesized using a standard Fmoc-based SPPS strategy (Scheme 1, details in SI).19,20 Fmoc-Ala-Wang resin is used for the solid support. The resin is swelled in dichloromethane (DCM) while synthesis steps utilize DMF as the solvent. Piperidine (20% in DMF) was used for the Fmocdeprotection steps, and the coupling steps are performed in 25% DIPEA in DMF. The coupling steps use 4 mol equiv of the Fmoc-protected amino acids and N,N,N′,N′-tetramethyl-O(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) with respect to the growing peptide. In the last coupling step, hexanoic acid is used in place of the amino acid to acylate the N-terminus. The peptide is cleaved from the resin using 95% trifluoroacetic acid (TFA) in water. The peptides are isolated following rotary evaporation of the TFA and precipitation in cold diethyl ether. The white solid is lyophilized from water to give the final product. Chemical characterization performed by B
dx.doi.org/10.1021/ed5001203 | J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Scheme 1. Overview of N-Hexyl-GAGA Synthesis Using SPPS Techniques: (a) Remove Fmoc Protecting Group; (b) Couple Fmoc-Protected Amino Acid; (c) Couple Hexanoic Acid to N-Terminus; (d) Cleave Peptide from Resin
Five of the 26 groups obtained products that contained impurities (one or more spots with lower Rf values on TLC), and two groups did not obtain any significant product (
