Activity pubs.acs.org/jchemeduc
Using Molecular Models To Show Steric Clash in Peptides: An Illustration of Two Disallowed Regions in the Ramachandran Diagram Christopher J. Halkides* Department of Chemistry and Biochemistry, The University of North Carolina at Wilmington, Wilmington, North Carolina 28403, United States S Supporting Information *
ABSTRACT: In this activity, students manipulate three-dimensional molecular models of the Ala-AlaAla tripeptide, where Ala is alanine. They rotate bonds to show that the pairs of dihedral angles ϕ = 0°, ψ = 180°, and ϕ = 0°, ψ = 0° lead to unfavorable interactions among the main chain atoms of the tripeptide. This activity allows the student to visualize in three dimensions why these two pairs of dihedral angles fall within the disallowed region of the Ramachandran diagram, a fundamental tool in the understanding and teaching of protein structure. The advanced activity introduces biochemistry students to unfavorable interactions of the β carbon of the side chain. KEYWORDS: Upper-Division Undergraduates, Biochemistry, Hands-On Learning/Manipulatives, Proteins/Peptides, Conformational Analysis
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The Ramachandran plot functions as a tool in the solutions of new protein structures and remains a subject of research. The Ramachandran diagram is used to find errors in refinement and has been incorporated into protein structure validation programs such as PROCHECK, MOLEMAN2, and MolProbity.5,6 Karplus and colleagues indicated the existence of conformation-dependent variation in the N−Cα−C bond angle greater than its standard value and also discussed small deviations of ω from 180°. These workers also suggested a standardized nomenclature and introduced the wrapped Ramachandran plot.6 Brasseur and colleagues brought electrostatic considerations into the conformational analysis and reconsidered the special cases of glycine and pre-proline residues, that is, residues on the N-terminal side of proline.3,7 Thus, there are at least four Ramachandran diagrams: for standard amino acids, for glycine, for proline, and for preproline.
n this activity, students manipulate a three-dimensional model of the Ala-Ala-Ala tripeptide (where Ala is alanine) and measure distances between atoms in two conformations. They learn about steric clashes that occur at certain dihedral angles. This activity helps students understand the basis of the Ramachandran diagram. The basic activity examines two steric clashes between main chain atoms, and the advanced activity examines two clashes that involve the side chain. The dihedral angles ϕ and ψ for the second residue in the Ala-Ala-Ala tripeptide are shown in Figure 1A. A Ramachandran diagram is a plot of allowed or disallowed values of the angles ϕ (abscissa) and ψ (ordinate) around the α carbon atom in a polypeptide backbone.1,2 A Ramachandran steric map emphasizing the particular atoms that clash in disallowed regions is shown in Figure 1B; a revised map has also been offered.3 Although ϕ and ψ may take on a variety of values, ω, the dihedral angle defined by the Cα−C−N−Cα atoms is limited to values near 180° for the trans configuration. Pauling, Corey, and Branson used the planarity of the peptide bond (Figure 2) as one of their constraints in proposing the structure for the α helix.4
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ABOUT THE ACTIVITY This classroom activity covers protein structure and is appropriate for an introductory course in biochemistry. The purpose of this activity is to show that two particular pairs of dihedral angle values in the Ramachandran each lead to steric clashes between two atoms. Students often have difficulty visualizing three-dimensional phenomena from two-dimensional representations. Therefore, teachers employ models as a means of experiential learning in both lecture and laboratory courses in organic chemistry to illustrate steric issues, such as the conformations of butane and cyclohexane. Although less frequently employed, models can also illustrate related concepts
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BACKGROUND The three-dimensional structure of the backbone of a protein is almost entirely specified by the values of ϕ and ψ for each residue, and the introduction to secondary structures in biochemistry courses is often closely intertwined with a discussion of the Ramachandran diagram. Therefore, the principles governing why some values of these two dihedral angles are unfavorable are the foundation of an understanding of protein structure. © XXXX American Chemical Society and Division of Chemical Education, Inc.
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Activity
produce at least 5 Ala-Ala-Ala tripeptide models, with the black tetrahedron being the limiting atom. The 3.5 cm straws are used for the single bonds (N−Cα and Cα−C), and the 3.0 cm straws are used for the partial double bonds (C−O and C−N). The C−O bonds, the C−N bonds, the N−Cα bonds, and the Cα−C bonds are 1.24, 1.32, 1.46, and 1.53 Å, respectively.9 The scale is 1 Å approximately equals 2.92 cm. These model kits include some trigonal carbon atoms with one bond angle of 108° and some trigonal nitrogen atoms with one bond angle of 114°. These atoms are not ideal for the representation of Cα and N, especially within Ala-2. However as suggested by the manufacturer, the Cα−C−N bond is slightly better represented with a trigonal carbon atom having a 114° angle than a 120° angle. The hydrogen atoms on the N-terminal amino group are usually omitted. Assembling a model takes about 15 min. The steric clashes between specific pairs of atoms determine the shape of the Ramachandran diagram.6 The models are laid out in the zig−zag conformation (ϕ = ψ = 180°) at the beginning of the activity, in which all of the main chain atoms lie in a plane and the methyl groups face on alternate sides of the plane (Figure 3A). Emphasizing the color scheme of the atoms helps the students to recognize the N- and C-termini. In the second, intermediate conformation, ϕ = 0°, ψ = 180°, the two oxygen atoms are no more than 4.6 cm apart, corresponding to 1.6 Å (Figure 3B). This value is much less than the extreme limit of 2.6 Å, which corresponds to about 6.1 cm.10 The distance in the model is roughly the length of the oxygen−oxygen single bond in hydrogen peroxide. The region near ϕ = ψ = 0° produces a steric clash between the carbonyl oxygen atom of residue i − 1 with the amide hydrogen (HN) of residue i + 1 (Figure 3C). Time permitting, the instructor inspects the models to see that the tripeptide model achieved the third, final conformation ϕ = 0, ψ = 0. Students often fail to realize that O of Ala-1 and HN of Ala-3 are too close to each other in the f inal conformation, ϕ = ψ = 0°. For the conformation ϕ = ψ = 0°, the oxygen and hydrogen atom are 1.0−1.8 cm apart for these models, which corresponds to 0.35−0.62 Å (Figure 3C). The actual distance in this conformation is 0.35 Å.10 An oxygen atom and a hydrogen atom that are not covalently bonded cannot be closer than 2.2 Å of each other, which corresponds to 6.4 cm. By way of further comparison, the distance between an oxygen atom that is covalently bonded to a hydrogen atom is about 0.96 Å, corresponding to 2.8 cm. The model incorporates some compromises with respect to bond lengths and angles,
Figure 1. Definitions of ϕ and ψ and their relationship to steric clash. (A) The dihedral angles ∠C−N−Cα−C and ∠N−Cα−C−N. (B) The original Ramachandran steric map1 where two of the hard-sphere repulsions (dashed lines) identified by Mandel et al.2 are shaded. The regions demarcated by Oi−1···O (purple) and Oi−1···Hi+1 (pink) are treated in this activity. Adapted with permission from ref 3. Copyright 2003 The Protein Society.
Figure 2. A peptide bond between two amino acid residues showing the definition of the dihedral angle ω. The six atoms of the peptide bond lie approximately in a plane. The leftmost Cα, C, and O atoms belong to residue i, and the rightmost N, Cα, and HN atoms belong to residue i + 1. The dihedral angle ∠ Cα−C−N−Cα is usually close to 180°.
in biochemistry and teach what one group termed “three dimensional molecular literacy”.8 Cochranes molecular models are a good choice on the basis of cost, ease-of-assembly, and ease of use (one space-filling model kit was not easy to assemble). One Cochranes molecular model kit for biochemistry (Sigma-Aldrich Z184764-1KT) will
Figure 3. (A) The Ala-Ala-Ala tripeptide in the zig−zag conformation, in which the values of all of the main chain dihedral angles are 180°. When ϕ = 180°, the trigonal black atoms representing carbonyl carbon atoms of Ala-1 and Ala-2 are furthest apart from each other. When ψ = 180°, the trigonal, blue atoms representing amide nitrogen atoms of Ala-2 and Ala-3 are furthest apart from each other. Oxygen atoms are red, hydrogen atoms are white, and the black, tetrahedral atoms are sp3-hybridized carbon atoms. (B) Rotation of ϕ to 0° produces strong repulsion between the oxygen atoms of Ala-1 and Ala-2 (red), which approach each other much more closely than two nonbonded oxygen atoms should. (C) When ϕ = 0°, subsequent rotation of ψ to 0° produces strong repulsion between O (Ala-1) and HN (Ala-3), which approach to 0.35 Å of each other, much less than even the length of a covalent bond. B
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ACKNOWLEDGMENTS I am very grateful to William Fridrich for providing detailed photographs. I am grateful to my colleagues and to the reviewers for many helpful suggestions.
and some model-to-model variation is observed, both of which limit the reliability of the distance estimates. The advanced activity is optional and is for those instructors who wish to explore the Ramachandran plot in more depth with their students, either as part of the classroom activity or as a homework assignment. All amino acid residues except glycine have a β carbon atom, and clashes with this atom occur in the bottom third and rightmost quarter of the Ramachandran plot. This activity illustrates some of these steric interactions. Students rotate ϕ to find the closest approach of Cβ of Ala-2 to O of Ala-1 and measure this distance. Students rotate ψ to find the closest approach of Cβ to HN, and they can measure these distances with a ruler. If rulers are unavailable, students can estimate these distances using their smallest finger or ring finger, which are about 6 cm (2.1 Å) and 7 cm (2.4 Å), respectively, when measured from the palm side. A number of interatomic distances as functions of ϕ or ψ have been plotted.3 The steric clashes involving Cβ are less obvious than the clashes between the main-chain atoms, but the distances are below the van der Waals diameters. Students can be questioned about which amino acid lacks these clashes.
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REFERENCES
(1) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. Stereochemistry of Polypeptide Chain Configurations. J. Mol. Biol. 1963, 7, 95−99. (2) Mandel, N.; Mandel, G.; Trus, B. L.; Rosenberg, J.; Carlson, G.; Dickerson, R. E. Tuna Cytochrome c at 2.0 Å Resolution. III. Coordinate Optimization and Comparison of Structures. J. Biol. Chem. 1977, 252, 4619−4636. (3) Ho, B. K.; Thomas, A.; Brasseur, R. Revisiting the Ramachandran Plot: Hard-Sphere Repulsion, Electrostatics, and H-Bonding in the αHelix. Protein Sci. 2003, 12, 2508−2522. (4) Pauling, L.; Corey, R. B.; Branson, H. R. The Structure of Proteins: Two Hydrogen-Bonded Helical Configurations of the Polypeptide Chain. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 205−211. (5) Kleywegt, G. J.; Jones, T. A. Phi/Psi-Chology: Ramachandran Revisited. Structure 1996, 4, 1395−1400. (6) Hollingsworth, S. A.; Karplus, P. A. A Fresh Look at the Ramachandran Plot and the Occurrence of Standard Structures in Proteins. Biomol. Concepts 2010, 1, 271−283. (7) Ho, B. K.; Brasseur. BMC Struct. Biol. 2005, 5, 14. (8) Bateman, R. C., Jr.; Booth, D.; Sirochman, R.; Richardson, J.; Richardson, D. Teaching and Assessing Three-Dimensional Molecular Literacy in Undergraduate Biochemistry. J. Chem. Educ. 2002, 79, 551−552. (9) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 5th ed.; W.H. Freeman and Company: New York, 2008, pp 121−122. (10) Ramachandran, G. N.; Sasisekharan, V. Conformation of Polypeptides and Proteins. Adv. Protein Chem. Struct. Biol. 1968, 23, 283−438. (11) Garrett, R. H.; Grisham, C. M. Biochemistry, 4th ed.; Brooks/ Cole: Boston, MA, 2010; pp 137−138.
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INTEGRATING THE ACTIVITY INTO THE CURRICULUM The Ramachandran plot is found in the chapter on protein structure in many introductory textbooks.9,11 Although some advanced textbooks use a form of this diagram that is an energy contour, a form often encountered is a classification of pairs of ϕ/ψ angles as allowed, allowed at extreme limits, permissible, and disallowed. The Ramachandran plot shows that the allowed values of ϕ and ψ are interdependent. Students are typically taught how the dihedral angle affects the energy of butane in their introductory organic chemistry course; however, the energy of butane is shown as being dependent upon on a single dihedral angle. Thus, the codependence of energy on two dihedral angles is an extension of a previously encountered concept. In the lectures leading up to the one in which the activity is performed, the instructor discusses the planarity of the peptide bond and its lack of free rotation. He or she may also introduce Newman projections or ball and stick drawings to illustrate the definitions, and some values, of the peptide chain dihedral angles ϕ and ψ. Immediately before the tutorial, he or she projects an image of the Ramachandran diagram and discusses the allowed and disallowed areas in terms of steric clashes and low versus high energy. After the tutorial, the students may refresh themselves on the concepts using photos of the model or a video of the demonstration.
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Activity
ASSOCIATED CONTENT
S Supporting Information *
A student activity worksheet. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. C
dx.doi.org/10.1021/ed3001528 | J. Chem. Educ. XXXX, XXX, XXX−XXX
