Peptide Structure: From Amino Acid Chain to Folded Shape

Peptide structure is described at several levels. The primary structure is the chain of amino acids itself, written from the N-terminus to the C-terminus. Secondary structure is the local shape of the backbone, mainly alpha helices, beta strands and turns, held by hydrogen bonds between backbone groups. Tertiary structure is the overall three-dimensional fold of one chain. Underneath all of it is the peptide bond, a planar amide link whose C-N bond (about 1.33 Å) is shorter than a normal single bond and does not rotate freely.

This article covers how a chain of amino acids is built and described, what controls its shape, how chemical modifications change it, and how a laboratory confirms the structure of a sample. For the building blocks themselves, start with what an amino acid is.

Peptide structure at a glance

Source: standard structural-biology usage; methods per references [5] and [11] to [14].

A chain of amino acids: the backbone

A peptide is a chain of amino acids joined head to tail. The amino group of each residue forms an amide bond with the carboxyl group of the residue before it, and one water molecule is released per bond. The repeating unit of the chain is N-Cα-C(=O), so the backbone reads N-Cα-C-N-Cα-C and so on, with each residue’s side chain branching off its alpha carbon.

Two ends are left over. One residue keeps a free amino group: that end is the N-terminus. The residue at the other end keeps a free carboxyl group: that end is the C-terminus. The two ends are chemically different, so a chain has a direction, the same way a word does. Peptide chains, whether short synthetic oligomers or long polypeptide chains, are always read in that direction.

The peptide bond: planar and mostly trans

The amide bond linking two residues is the single most important structural feature of a peptide. The nitrogen’s lone pair is shared with the neighbouring carbonyl group (resonance), giving the C-N bond partial double-bond character. In practice this means:

• The bond is short. At about 1.33 Å, the peptide C-N bond sits between a typical C-N single bond (about 1.47 Å) and a C=N double bond (about 1.27 Å).
• The group is flat. The carbonyl carbon, its oxygen, the amide nitrogen, its hydrogen and the two flanking alpha carbons lie close to one plane. Pauling, Corey and Branson built this planar amide into their 1951 models of the polypeptide chain, which led to two hydrogen-bonded helical configurations [1].
• Rotation is restricted. The angle around the C-N bond, called omega, stays near 180° (trans) or, rarely, near 0° (cis).

Trans is strongly preferred because the cis form brings the two neighbouring alpha carbons into contact. The exception is the bond in front of proline. Stewart, Sarkar and Wampler surveyed the Brookhaven Protein Data Bank in 1990 and found that only 17 of 31,005 ordinary amide bonds (0.05%) were cis, compared with 99 of 1,534 bonds preceding proline (6.5%). The cis bonds they found sat mainly in bends and turns [2]. Cis-trans switching in front of proline is slow on a laboratory timescale, which is one reason a proline-containing peptide can show more than one set of signals in NMR.

Backbone angles and the Ramachandran plot

With omega held near 180°, the shape of a chain comes down to two rotations per residue: phi (around the N-Cα bond) and psi (around the Cα-C bond). Ramachandran, Ramakrishnan and Sasisekharan worked out in 1963 which combinations of phi and psi are possible without atoms bumping into each other [3]. Plotted as a map, the allowed combinations form a few islands. The largest correspond to the alpha helix and the extended beta strand.

Side chains change the map. Glycine has only a hydrogen as its side chain, so it can reach regions closed to every other residue, which is why glycine is common in tight turns. Proline’s ring locks phi near -65°, so it fits poorly inside a helix and often sits at the start of one or in a turn. These two residues are the main reason sequence and shape are so closely linked.

Secondary structure: helices, sheets and turns

The alpha helix

In an alpha helix the backbone coils so that the C=O of residue i hydrogen-bonds to the N-H of residue i+4. There are about 3.6 residues per turn, each residue advances the helix by about 1.5 Å, and one full turn rises about 5.4 Å. The side chains point outward. Pauling, Corey and Branson proposed this configuration in 1951 from the geometry of the planar amide [1], before any protein structure had been solved experimentally.

The beta strand and beta sheet

A beta strand is an almost fully extended stretch of chain, about 3.3 to 3.5 Å per residue. Two or more strands lie side by side and hydrogen-bond to each other to form a sheet, either in the same direction (parallel) or alternating (antiparallel). Because each residue’s plane tilts slightly, the sheet has a pleated look, which gave the structure its name in Pauling and Corey’s 1951 paper [4].

Turns and loops

Turns reverse the direction of the chain over a few residues. The common beta turn spans four residues and is closed by a hydrogen bond between the first and the fourth. Glycine and proline are over-represented in turns for the backbone-angle reasons above, and cis bonds before proline cluster here too [2].

Assigning secondary structure from a set of atomic coordinates is not a matter of judgement. Kabsch and Sander published a standard method in 1983, the Dictionary of Secondary Structure of Proteins (DSSP), which labels each residue from its hydrogen-bond pattern and geometry [5]. DSSP-style labels are what most structure databases display.

Tertiary structure: how a chain folds

Tertiary structure is the full three-dimensional arrangement of one chain: which helices and strands pack against which, and where each side chain sits. Water-avoiding side chains tend to cluster away from water, polar and charged ones toward it, and disulfide bridges, hydrogen bonds and salt bridges lock the arrangement in place.

Anfinsen summarised the central principle in 1973: for the proteins his group studied, the native three-dimensional structure is set by the amino acid sequence under a given set of conditions, because the folded form is the one of lowest free energy [6]. That is the reason primary structure matters so much. Change the sequence, and the shape the chain can adopt changes with it.

Short peptides are different. A chain of 5 to 20 residues usually has too few internal contacts to hold a single fold, so in water it moves between many conformations. Some short sequences do favour a helix or a turn, especially when cyclised or bridged by a disulfide. When several separate chains assemble into one unit, as in many larger proteins, the arrangement of the chains is called quaternary structure.

How peptide sequence is written

The written form of a peptide is its primary structure, and it follows IUPAC-IUB conventions [7]:

• The N-terminus is on the left and the C-terminus on the right. Ala-Gly and Gly-Ala are different compounds.
• Residues are written as three-letter codes joined by hyphens (Gly-His-Lys) or as a string of one-letter codes (GHK).
• L residues are assumed. A D residue is marked with a D- prefix.
• Free ends can be shown as H- (amine) and -OH (acid). Modified ends take a prefix or suffix, such as Ac- or -NH2.
• Rings and bridges are stated explicitly: cyclo(…) for a head-to-tail ring, and the paired positions for each disulfide.

Numbering also runs N to C, starting at 1. When a lab report or paper refers to “position 5”, it means the fifth residue counted from the N-terminus. Our primer on what peptides are gives more examples of the notation.

Peptide modifications that change structure

Many peptides are more than a plain chain. Each modification below changes the molecular formula, so each one is visible to mass spectrometry.

• C-terminal amidation replaces the terminal -COOH with -CONH2 (about -0.98 Da). It removes the negative charge at that end. In cells, the amide is made from a glycine-extended precursor by the copper enzyme peptidylglycine alpha-amidating monooxygenase [8]. In synthesis it is usually built in by assembling the chain on an amide-forming resin.
• N-terminal acetylation adds CH3CO- to the free amine (+42.01 Da) and removes the positive charge at that end. It is a widespread modification of natural proteins in both eukaryotes and prokaryotes [9].
• Cyclisation joins the two ends, or a side chain to an end or to another side chain. Ring closure restricts the conformations the chain can adopt, and forming the ring is a known synthetic challenge [10]. A head-to-tail ring loses one water (-18.01 Da).
• Disulfide bridges link two cysteine side chains (-S-S-), each bridge removing two hydrogens (-2.02 Da). They can tie one chain into a loop or join two chains.

Modifications add structure that the sequence letters alone do not show. That is why a complete identity statement for a peptide names the sequence, the end groups, any rings or bridges, and any counter-ion. How these features are introduced during manufacture is covered in how peptides are made.

How peptide structure is confirmed

Each level of structure has its own analytical tools.

Primary structure

• Intact mass. Mass spectrometry measures the mass of the whole molecule and compares it with the value calculated from the sequence and modifications. A missing residue, an unformed disulfide or a lost amide all shift the mass by a known amount.
• Tandem MS. The peptide ion is broken along its backbone, mostly at the amide bonds, giving two ladders of fragments read from either end. Steen and Mann described how these fragment series are read back into a sequence [11]. Tandem MS confirms order, which intact mass cannot.
• Edman sequencing. The N-terminal residue is chemically removed and identified, one at a time. Edman and Begg automated the process in 1967 with their protein sequenator [12]. It is slower than MS but reads sequence directly from the N-terminus, and it cannot start if the N-terminus is blocked, for example by acetylation.

Secondary and tertiary structure

• Circular dichroism (CD). Helices, sheets and flexible chains each give a characteristic far-UV CD spectrum. Greenfield’s methods paper describes how spectra are used to estimate secondary-structure content, and notes that CD does not give the residue-specific detail of crystallography or NMR [13].
• NMR spectroscopy. Solution NMR measures distances and angles between specific atoms and turns them into a three-dimensional model. Wüthrich, whose group developed the approach, gave a first-hand account of how protein structure determination by NMR came about [14]. It suits short peptides because it captures the range of conformations present in solution.
• X-ray crystallography gives an atomic model of a molecule in a crystal, from which DSSP-style secondary-structure labels can be assigned [5].

For a lyophilised research peptide, the routine checks on a lab report are intact mass (identity) and HPLC (purity). Higher-order methods like CD and NMR are research measurements rather than lot-release tests. Our explainers on reading a peptide COA and HPLC peptide purity show how those two results are read together.

Frequently asked questions

What is the structure of a peptide?

A peptide is a chain of amino acid residues joined by planar amide (peptide) bonds, with a free amino group at the N-terminus and a free carboxyl group at the C-terminus. Its primary structure is that sequence. Depending on length and sequence, the chain can also form local helices, strands or turns.

Why is the peptide bond planar?

The nitrogen lone pair is shared with the adjacent carbonyl group, which gives the C-N bond partial double-bond character. That keeps the six atoms around the bond close to one plane and restricts rotation. The bond is almost always trans, except that cis is noticeably more common in front of proline.

Why are peptides written from the N-terminus to the C-terminus?

The two ends of a chain are chemically different, so a sequence has a direction. IUPAC-IUB convention writes the free amino end on the left and numbers residues from there. Reversing the order describes a different compound.

How is the structure of a peptide confirmed?

Mass spectrometry confirms the intact mass and tandem MS confirms residue order. Edman sequencing reads residues from the N-terminus. Circular dichroism and NMR describe secondary and three-dimensional structure in solution.

Research materials: Vortex supplies peptides as sealed lyophilised research reagents. Browse the catalogue at /shop/. Published lab reports are at /coa/.

References

1. Pauling L, Corey RB, Branson HR. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci U S A. 1951;37(4):205-11. PMID 14816373. doi:10.1073/pnas.37.4.205
2. Stewart DE, Sarkar A, Wampler JE. Occurrence and role of cis peptide bonds in protein structures. J Mol Biol. 1990;214(1):253-60. PMID 2370664. doi:10.1016/0022-2836(90)90159-J
3. Ramachandran GN, Ramakrishnan C, Sasisekharan V. Stereochemistry of polypeptide chain configurations. J Mol Biol. 1963;7:95-9. PMID 13990617. doi:10.1016/s0022-2836(63)80023-6
4. Pauling L, Corey RB. The pleated sheet, a new layer configuration of polypeptide chains. Proc Natl Acad Sci U S A. 1951;37(5):251-6. PMID 14834147. doi:10.1073/pnas.37.5.251
5. Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22(12):2577-637. PMID 6667333. doi:10.1002/bip.360221211
6. Anfinsen CB. Principles that govern the folding of protein chains. Science. 1973;181(4096):223-30. PMID 4124164. doi:10.1126/science.181.4096.223
7. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature and symbolism for amino acids and peptides. Recommendations 1983. Eur J Biochem. 1984;138(1):9-37. PMID 6692818. doi:10.1111/j.1432-1033.1984.tb07877.x
8. Prigge ST, Mains RE, Eipper BA, Amzel LM. New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci. 2000;57(8-9):1236-59. PMID 11028916. doi:10.1007/pl00000763
9. Ree R, Varland S, Arnesen T. Spotlight on protein N-terminal acetylation. Exp Mol Med. 2018;50(7):1-13. PMID 30054468. doi:10.1038/s12276-018-0116-z
10. White CJ, Yudin AK. Contemporary strategies for peptide macrocyclization. Nat Chem. 2011;3(7):509-24. PMID 21697871. doi:10.1038/nchem.1062
11. Steen H, Mann M. The ABC’s (and XYZ’s) of peptide sequencing. Nat Rev Mol Cell Biol. 2004;5(9):699-711. PMID 15340378. doi:10.1038/nrm1468
12. Edman P, Begg G. A protein sequenator. Eur J Biochem. 1967;1(1):80-91. PMID 6059350. doi:10.1007/978-3-662-25813-2_14
13. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006;1(6):2876-90. PMID 17406547. doi:10.1038/nprot.2006.202
14. Wüthrich K. The way to NMR structures of proteins. Nat Struct Biol. 2001;8(11):923-5. PMID 11685234. doi:10.1038/nsb1101-923

Methods note: written by the Vortex Research Editorial Team. Literature was selected from PubMed for chemistry, structural and analytical topics, every PMID was checked against its PubMed record for title, journal and year, and each abstract (where PubMed holds one) was read against the sentence that cites it. Mass differences were calculated from atomic composition.

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