MODULE 23

Analysis of Macromolecular structure - Ramachandran Plot

Learning Objectives

 Protein structure classification  Conformation of a protein  Understanding the Ramachandran Plot  Validation of a protein structure using Ramachandran Plot

23.1. Role of proteins in biology The proteins are functional tool of living organism, plays vital role in cell functions such as structural support, defense against pathogens, transportation along the cell walls, etc.

23.1.1. Protein Proteins are composed of twenty different amino acids with different chemical composition of nitrogen, carbon, hydrogen and oxygen. The function of each protein directly related to structure of the protein. To attain particular structure, proteins were synthesized using twenty different amino acids having different chemical structure and characteristics. Thus function of a protein varies with structure, varies with the composition of amino acids and the sequence complexity.

23.1.2. Protein structure classification Structure a proteincan be classified in to four classes; primary, secondary, tertiary and quaternary structure by degree of structural complexity. Primary structure is amino acid sequence that creates polypeptide chain. In Secondary structure, non-covalent interactions particularly hydrogen bonds between amino acids form preliminary three-dimensional structures such as α-helices and β-strands. Tertiary structure describes the assembly of secondary structures to obtain the overall structure of the protein, mainly divided into globular and fibrous proteins. Quaternary structure explains the three-dimensional structure representation of a protein having two or more polypeptide chains linked by di-sulfide bridges or hydrogen bonds.

23.2. Conformation of a protein

The spatial arrangement of atoms in a given molecules is described as conformation which is true also for proteins. Conformation explains not only the spatial arrangement but also the possibility of

assuming other possible orientations. This change is not via breaking covalent bond but mere rotating a bond that connects atoms together. This conformation changeability of a protein helps for exhibiting a function such as like on/off switch to function or adaptability upon approach of a ligand which may a small or macromolecule. The stable, folded, functional conformations are called native protein. The studies on conformation of a protein give the understanding towards the functional mechanism of a protein.

The X-Ray diffraction experiment used to get the possible stable conformation of a protein. From the diffraction experiment, researchers used to build protein structures. Ramachandran plot or ϕ, ψ plotis serving as a tool to validate the arrived model from any experimental technique and remained nearly unchanged in the past fifty years and continues to be an integral part of structural biology.

From the above figure one can understand that the Phi and Psi are torsional angles about N-Cα and Cα-C’ bonds by which the two adjacent peptide units can be oriented.

23.3. Ramachandran plot in structure refinement Since structure determination by X-ray Crystallography (about 8000 PDB entries) and NMR became routine, usage of this plot is also increased to assess the quality of the structure.

These are the frequent questions put forward to a crystallographer. 1. Is the crystallographic model (position of atoms) is justified? 2. Since the enzymes or proteins perform functions by dynamic motion of atoms, does crystallography give us any insight into these motions? 3. Are some parts of the molecule more flexible than others? 4. Are major movements of structural elements essential to the molecule's action?

When the last three questions can be answered from thermal factor analysis for individual amino acids, the first question demands the fundamental validation.

23.4. How does Ramachandran plot validate the protein conformation? 23.4.1. Steric clashes In space, at any instance, atoms cannot occupy same space. And covalent bond cannot be broken without any energy transformation. There possibility of rotation and clashes may occur between atoms and only two possible degrees of rotation in amide backbone. From the above knowledge, Prof. G. N. Ramachandran made three strategies to find steric clashes in mono and dipeptide.

Strategy I Set φ to zero and vary ψ

ψ is near 0°, O1-H2 clash

ψ is near 180° O1-O2 clash

At any value of ψ O1-C2clashes are observed at φ= -45 to 45

Strategy II Set ψ to zero and vary φ

φ is near 0° O1-H2 clash

φ is near 180° H1-H2 clash

At any value of φ N1-H2 clash Ψ = -20 to 20

Overall backbone- backbone interaction by varying φ and ψ.

Strategy III

23.4.2. Backbone – side chain clashes

To search clashes between side chain and backbone the ψ was varied. And it found to be a steric clash between R-H2 from the range of ψ = -65° to ψ = -175°. By varring φ the O1-R steric clash was observed between range from φ = 65° to φ = 175.

overall backbone- side chain interaction by varying φ and ψ.

23.5. The Ramachandran Plot From above findings, Prof.G.N.Ramachandran developed the following plot to describe allowedφand ψ combinations between amide planes

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From the analysis of existing crystal structures available at that time the preference of phi-psi combination by secondary structure components were assigned.

The following figure explains the good and bad model with relevance to the Ramachandran outliers.

Ramachandran Plot is used to guide for a proper structure refinement.

The above image depicts initial (R = 49 %) and final (R = 18 %) structure refinement where number of outlier reduced drastically (Divneetal.,) cellobiose hydrolase.

23.6. Unique Features of Ramachandran plot in Structural analysis

1. The Ramachandran plot clearly depicts how well the phi and psi angles cluster and reveals other oddities that may be the result of errors made during refinement. 2. Ramachandran plot is one of the simplestand most sensitive means for assessing the quality of aprotein model in the absence of experimental data since the φ and ψ angles (or combinations of these) are not usually restrained during X-ray refinement, as opposed to bond lengths and bond angles, for instance. 3. Crystallographic software packages and map-fittingprograms usually contain a routine for computingφ and ψ for each residue from the current coordinate list, as well as for generating the Ramachandran diagram and plotting a symbol or residue number at the position (φ,ψ).

23.7. Some Good Things about Outliers

When an outlier warns some discrepancy, sometimes there could be a reason for such anomaly. In-fact such disagreement may occur as a response to an input signal such as ligand binding. As a consequence such conformational changes could contribute change in enzyme activity or in the affinity of protein-ligand, protein-protein or protein-DNA interactions (West et al,.).

Summary

 Hence structure determines the function ofa protein, to arrive a correct model of the structure is very important  Ramachandran Plot is highly useful to validate an experimentally determined structure.  The plot explains the possible orientation (energetically allowed) of two successive peptide unit with relevance to the central Cα carbon atom.  Apart from this, the plot is highly useful to monitor the progress of a successful structure refinement.  Hence in all the structure refinement softwares, the plot is incorporated to see the real time movement of phi psi combination upon changes in orientation of a given amino acid.  The unavoidable outlier in a plot may contribute for the function by of a ligand or by exhibiting a molecular switch kind of arrangement.