4.1: Main Chain Conformations

Search Fundamentals of Biochemistry

Learning Goals (ChaptGPT o1, 1/25/25)

• Understand Basic Conformational Principles:

  • Explain the concept of conformers using simple molecules (e.g., butane) and interpret Newman projections to compare energy differences between staggered (anti) and eclipsed (syn) conformations.
  • Relate the idea of dihedral angles in small hydrocarbons to the rotation around bonds in longer chains, such as in dodecanoic acid and peptide backbones.

• Connect Simple Organic Conformations to Peptide Structure:

  • Compare the extended, low-energy “all-trans” (anti) conformation in nonpolar chains with the typical zig-zag arrangement found in protein backbones.
  • Describe how dihedral angles (phi and psi) dictate the conformation of the peptide main chain, and distinguish between the conformations typical of beta sheets (near 180°) and alpha helices (approximately 60°).

• Explore Peptide Bond Isomerism and Its Consequences:

  • Differentiate between trans and cis peptide bonds and explain why the trans configuration is highly favored (except in X-Pro bonds) due to reduced steric hindrance and torsional strain.
  • Analyze how the presence of a cyclic amino acid like proline affects the cis/trans ratio in peptide bonds.

• Master Backbone Dihedral Angles and Ramachandran Plots:

  • Define the phi (φ) and psi (ψ) dihedral angles and explain their significance in determining the overall backbone conformation.
  • Interpret Ramachandran plots to identify allowed and disallowed regions of φ/ψ space, and relate these regions to common secondary structures (alpha helices, beta sheets, and turns).
  • Discuss how the planarity of the peptide bond (due to resonance) restricts the conformational freedom of the protein backbone.

• Apply Computational and Experimental Approaches:

  • Understand how experimental models (e.g., interactive iCn3D models) and computational tools (e.g., force fields using Lennard-Jones potentials) aid in visualizing and predicting backbone conformations.
  • Recognize the importance of considering both steric and electronic factors in modeling peptide conformations and their implications for protein folding and function.

These learning goals will enable students to integrate fundamental organic chemistry concepts with protein structure analysis, providing a strong foundation for understanding how backbone conformation influences the secondary, tertiary, and quaternary structures of proteins.

Introduction

In Chapter section 3.2, we discussed primary, secondary, tertiary, and quaternary structures of proteins. We used the protein hydroxynitrile lyase as an example (5Y02). An interactive iCn3D model of hydroxynitrile lyase is shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): hydroxynitrile lyase (5Y02). Click the image for a popup or use this external link:
https://structure.ncbi.nlm.nih.gov/icn3d/share.html?gGY8QBkCP8dPmfoH6 (Copyright; author via source)

Blue indicates random coil, red indicates alpha helices, and yellow shows beta strands. As discussed in Chapter 3.2, the beta-strand backbones are quite extended compared to the alpha helices, which are more compressed. Atomic force microscopy can be used to pull the helix into alternative extended shapes. Let's explore main chain conformations in greater detail.

If we wish to understand protein structure, it is essential first to understand the possible conformations of the backbone chain before we start considering the effect of side chains and the overall structure of the protein. Where do we start? It's a highly complex topic, but we can simplify it by considering the structures of short hydrocarbons and other organic groups and using ideas you learned in earlier chemistry courses. So, let's start with butane, a 4C hydrocarbon.

You will remember from previous chemistry courses that different rotational conformations (conformers) of butane have different energies. These conformers are often visualized using Newman projections, which are sighted down the internal C2-C3 carbon bond in butane. The figure below shows ball and stick representations, and Newman projects for three different conformers formed by rotation around the C2-C3 bond. You are familiar with bond angles, the angle between two bonds connecting three connected atoms. For this discussion, we will consider the dihedral angle, which describes the rotation angle around the middle bond of a 4-atom, 3-bond connected system. It's easiest to use Newman projections to understand dihedrals, representing the angle between groups bonded to the front and back (circle) atoms sighted down the front-to-back atoms. Figure \(\PageIndex{2}\) shows multiple conformers of butane, represented in ball and stick form or as Newman projections.

Figure \(\PageIndex{2}\): Newman projections for butane

The syn and eclipsed conformer with a dihedral angle of 0° has the highest energy since the two eclipsed bonds have high torsional energy and the two closest approaching H atoms on the methyl C atoms have steric (1,6) interactions. Rotating the front C 60° gives a gauche conformation of lower energy. Rotating to 120° leads to higher energy, given the torsional strain between the eclipsed bonds. Rotating fully to 180° gives the conformer of lowest energy and torsional (bond-bond) strain. The steric strain between the two closest H atoms on the methyl groups also raises the energy.

A red line has been drawn between the four carbon atoms connected by three bonds in the anti or trans-conformer. Notice that the red line is a zig-zag, as shown in the rotated red line drawing beneath the ball and stick conformer. All the conformers are available, but the anti or trans-conformer is the most stable and abundant.

Let's try this approach on a more complicated molecule, a 12-carbon atom carboxylic acid, dodecanoic acid. Figure \(\PageIndex{3}\) shows the molecule in the all- trans conformation (where all the carbon atoms in the chain are arranged in an anti, zig-zag manner), and a second gauche conformation for the bond highlighted by a red line. Note that rotation around that bond to produce the gauche conformer, a higher energy form, produces a kink in the chain. We will see this again when we discuss lipid structure.

Figure \(\PageIndex{3}\): Conformations of dodecanoic acid

Now let's compare the all-trans dodecanoic acid structure to one largely extended conformer of a tetrapeptide, Gly-Gly-Ala-Gly (GGAG), as shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Extended conformations of dodecanoic acids and a tetrapeptide

Notice the similarities in these two structures. The atoms in the main chain of the peptide, N, C_α, C (the carbonyl C), and the carbonyl O, are arranged in the familiar zig-zag fashion, characteristic of the lowest energy conformer. The dihedral angles along the backbone would be, to a first approximation, around + 180° depending on whether the rotation is clockwise or counterclockwise from the syn conformer. However, the actual conformation of the GGAG peptide, if found as part of the primary structure of a protein, could adopt a whole range of other conformers with potentially different dihedral angles for each bond in the main chain. Look back at the iCn3D model for hydroxynitrile lyase above, and it should be clear that if the stretch of amino acids Gly-Gly-Ala-Gly is in a beta sheet, its dihedral angles for the main chain would be closer to +180⁰ than if they were in an alpha helix, which would dihedral angles much less than 180°, perhaps closer to + 60⁰

It might seem like all dihedral angles are possible for the main chain atoms of a protein, which would make understanding protein structure too complicated for our minds to comprehend. However, two significant factors simplify conformational analyses of proteins.

Trans and Cis Peptide Bonds

In the extended peptide example above, the alpha Cs of adjacent amino acids are on opposite sides of the peptide bond between the carbonyl carbon atom and the amide nitrogen atom. They are trans to that bond. A rectangle was drawn on the above figure centered on the C-N peptide bond for two adjacent amino acids to show the trans orientation of the alpha Cs.

Rotation around single bonds can occur, so an isomerization of the trans peptide bond to the cis isomer can occur. Both isomeric forms of the dipeptide Ala-Ala are shown in the top part of Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Cis/Trans peptide bonds

This trans arrangement of the alpha Cs in Ala-Ala and most internal X-Y amino acids pairs in a protein is sterically favored by a factor of 1000/1 over the cis form, which clearly shows torsional between the aligned (red) bonds and steric strain between amino acid side chain atoms.

The case is different for X-Y amino acid pairs when Y is proline, as shown in the bottom part of the above figure. Proline is a cyclic amino acid and, as such, would be expected to be more sterically restricted in a protein sequence. In X-Pro peptide bonds in proteins, the trans/cis ratio is about 4/1. The torsional (between bonds) and steric strain (between atoms) are similar in both isomers. You would expect to find both trans and cis X-Pro peptide bonds in proteins, given the constraints placed on protein conformation in the tertiary structure of the protein.

Figure \(\PageIndex{6}\) is an interactive iCn3D model showing two pairs of X-Y peptide bonds in the protein carboxypeptidase. Answer the questions below about the two peptide bond pairs, shown in stick form and CPK colors.

Figure \(\PageIndex{6}\): 2 peptide bonds in carboxypeptidase. Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3ARF97MgN4427A (Copyright; author via source)

Exercise \(\PageIndex{1}\)

Two X-Y amino acids are shown in stick form in the protein carboxypeptidase A

1. What is the sequence of each pair shown from N to C terminus

2. Are the X-Y peptide bonds cis or pro?

Answer

Add texts here. Do not delete this text first.

Backbone dihedral angles and Ramachandran Plots

Let's focus on possible dihedral angles for the peptide bond in protein chains. Just as saturated fatty acid chains have preferred conformations (all anti or trans), peptide chains also have preferred conformations. The complexity is much greater, however. For our dodecanoic acid example, we dealt only with torsion or dihedral angles around the methylene (-CH₂-) carbons. For proteins, we must consider the covalent links that attach the amino acids and the rotations possible in 20 different amino acids. The peptide bond connects the carbonyl C of the i^th amino acid to the alpha amine N of the i th+1 amino acid. The resulting bond is an amide link. X-ray analysis shows that the C-N bond has double bond character. This can be accounted for by delocalizing the nonbonding electron pair of the N to the carbonyl C, forming a double bond, with the pi-bonded electrons of the carbonyl C-O bond moving to the O. These resonance structures lead to a planar arrangement of the peptide carbonyl C and amide N and the two other atoms connected to each, since the hybridization of the C and N has sp² character, with 120^o bond angles, as illustrated in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Resonance forms of peptide bond

This greatly simplifies the number of conformations a protein can adopt since these six atoms can be considered to reside and move in a plane. The alpha C is the corner attachment point of two different planes, which can rotate independently of the other. The two planes can twist around the alpha carbon. The rotation angles for the two planes are called phi (φ), and psi (ψ) are analogous to the torsion angles in the acyl chain of dodecanoic acid. They can vary from -180 to +180^o. The R group substituent attached to the alpha C can also rotate around the side chain's alpha C and the beta C. This angle is defined as chi. Other rotations also occur within the side chain. We will concentrate on phi (φ) and psi(ψ) angles.

Figure \(\PageIndex{8}\) shows the peptide Gly-Gly-Ala-Gly (at high pH to give the protonation state shown) with the six atoms around the C-N peptide bonds shown in rectangles. Phi (φ) and psi (ψ) angles are also shown.

Figure \(\PageIndex{8}\): Phi/Psi angles for Gly-Gly-Ala-Gly

Using these ideas, we can consider a protein backbone a series of linked sequences of rigid, planar peptide units that rotate around phi/psi angles. When the chain is fully extended (as shown in the links above), φ/ψ are + 180^o, with the syn eclipse form defined as having 0⁰ φ/ψ angles. When φ and ψ equal 0^o, the two peptide bonds flanking the alpha Cs are in the same plane. This conformation is prohibited since the O of the C=O on one plane and the H of the H-N on the other overlap - i.e., they approach closer than their van der Waals radii.

The Proteopedia page below gives a phenomenal explanation and visual representation of φ/ψ angles in proteins.

Proteopedia: Ramachandran principle and phi psi angles

Figure \(\PageIndex{9}\) is an animated gif showing rotation of phi and psi angles (made from Proteopedia link above). You can see that when either φ or ψ approach 0⁰, steric clashes are most significant.

Figure \(\PageIndex{9}\): Animation of rotation around Phi/Psi angles f

Ramachandran was the first to calculate which φ/ψ angles are allowed. He modeled the angles permitted to a tripeptide, assuming the atoms were hard spheres. The angles allowed depended in part on the limiting distance chosen for interatomic contacts. (i.e., the usual H -- H distance is 2.0 Å and 3.0 Å for C--C bonds.) The plot below shows the allowed regions in red. Only three small regions of conformational space are available. If you allow a closer approach by 0.1 angstroms, more conformation space is available, but only one new area is available, shown in yellow in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Ramachandran plot. http://www.cryst.bbk.ac.uk/PPS95/cou...etry/rama.html

Right-hand alpha helices fall at -570,-470, while left-hand alpha helices fall at +570,+470. (Notice these are not mirror images of each other. The mirror image of a right-handed alpha helix would be a left-handed helix made of D-amino acids.) Parallel beta sheets are at -119⁰, -113⁰, while antiparallel sheets fall at -139⁰, +135⁰. Note that beta sheets have φ/ψ angles closer to the fully extended + 180⁰ of the all anti (trans) conformation of dodecanoic acid than the more compact alpha helix. Other types of helices also are found. The 310 helix, a sharper helix with 3 amino acids/turn, falls at -49⁰,-26⁰. All of these examples of secondary structure fall in allowed regions.

Modern Ramachandran plots do not model the atoms as hard spheres but use the potential energy of the atoms using the Lennard-Jones potential (6-12 potential) for induced dipole-induced dipole interactions. We discussed this potential function in Chapter 2.4.

A Ramachandran plot of Ala-Ala-Ala is nearly identical to the plot for Phe-Phe-Phe (which is unbranched at the beta carbon, the first methylene C in the side chain). The plot for Thr-Thr-Thr, which has a branch at the beta C (with OH and CH₃ attached), shows somewhat less room than the other plots. Pro-Pro-Pro is most restricted for obvious reasons.

For a longer chain than a tripeptide, there are more restrictions than for (Ala)₃ since the chain can't assume a conformation when it passes through itself. The plots for actual proteins have many points that fall in forbidden regions. However, these points would be allowed if the peptide bonds twist a few degrees. Gly bonds also fall outside the allowed areas. This is understandable since the side chain of Gly is H, and it is used in proteins where sharp turns of the chain are necessary.

Figure \(\PageIndex{11}\) shows Ramachandran plots for Gly, Ala, Tyr, and Pro in actual proteins (made years OK but can't find reference).

Figure \(\PageIndex{11}\): Ramachandran plots for Gly, Ala, Tyr, and Pro from actual proteins

Figure \(\PageIndex{12}\), taken from the Proteopedia page above, shows the Ramachandran plots for 100,000 proteins from the Protein Data Bank.

Figure \(\PageIndex{12}\): Ramachandran plots for 100,000 proteins. https://proteopedia.org/wiki/index.p...neral_100K.jpg

It's time to further explore secondary, tertiary, and quaternary structures. There are many ways to organize the structural organization of the protein world. For the rest of this chapter, we will adopt Carl Branden and John Tooze's approach in their seminal book, "Introduction to Protein Structure."

Summary

Chapter Summary

This chapter delves into the fundamental principles that govern the conformational properties of protein backbones—a critical aspect of understanding protein folding and structure. It begins by establishing the basic concepts of molecular conformation using simple organic molecules such as butane. By utilizing Newman projections, the chapter illustrates how rotational isomers—such as the high-energy eclipsed conformer and the low-energy anti (trans) conformer—result from variations in dihedral angles.

Building on this foundation, the discussion extends to more complex molecules like dodecanoic acid, where the concept of "all-trans" conformations is introduced, and kinks created by gauche rotations are highlighted. These ideas are then applied to peptide backbones, exemplified by a tetrapeptide (Gly-Gly-Ala-Gly), demonstrating that the peptide main chain adopts a similar zig-zag, extended conformation in its low-energy state.

The chapter further explains that peptide bonds, due to their partial double-bond character from resonance, are planar and predominantly adopt a trans configuration—except in cases involving proline, where the cis form is relatively more common. This planarity restricts the degrees of freedom for backbone rotations to two key dihedral angles, phi (φ) and psi (ψ), which define the orientation of adjacent peptide planes.

The chapter introduces Ramachandran plots, which map the allowed and disallowed regions of φ/ψ angles based on steric hindrance and energy considerations, to visualize and analyze these constraints. The plots reveal distinct clusters corresponding to common secondary structures: alpha helices, beta sheets, and random coils, and they underscore the unique conformational flexibility of residues like glycine and the restrictions imposed by proline.

Finally, the chapter sets the stage for deeper exploration of protein secondary, tertiary, and quaternary structures by adopting the framework outlined in classic texts like "Introduction to Protein Structure" by Branden and Tooze. Overall, this chapter highlights how understanding the backbone conformational landscape is essential for deciphering the complex three-dimensional architecture and function of proteins.