GENERAL ARTICLE Protein Structure - ias

GENERAL ARTICLE

Protein Structure ∗

Lalitha Guruprasad

Lalitha Guruprasad is aProfessor of Chemistry at the

School of Chemistry,University of Hyderabad,Hyderabad. Her research

interests are in understandingthe protein sequence tostructure and function

correlation.

Proteins are essential biological macromolecules associatedwith a variety of physiological functions. In this article, thecomposition, conformational features, and general principlesthat govern protein folding are presented.

1. Introduction

Proteins are essential building blocks of life. They are linearheteromeric biopolymers constructed from monomeric units (L-amino acids) covalently linked via an amide bond. There aretwenty naturally occurring amino acids. The chemical proper-ties of amino acids can be classified as polar (neutral, negativelyand positively charged), and non-polar (aliphatic and aromatic)based on the structure of the variable side chains (R). While thebackbone of a protein is monotonous with the repeating unit (seeinset in Figure 1), the variability in the chemistry of side chain‘R’ group makes the studies on protein structure exciting. Theamino acid sequence of a protein is the order in which the aminoacids are written, wherein, the first amino acid has the free amino(N–), and the last amino acid has the free carboxy (C–) terminalends.

2. Central Dogma

According to the central dogma of molecular biology, the flow ofgenetic information occurs from the deoxyribonucleic acid (DNA)to the ribonucleic acid (RNA) to the proteins. These processes Keywords

Proteins, amino acids, α-helix,

β-strand, protein folding, tertiary

structure.

are termed transcription and translation, respectively. Three nu-cleotides (one codon) taken at a time codes for one amino acid.

∗DOI: https://doi.org/10.1007/s12045-019-0783-7

RESONANCE | March 2019 327

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Figure 1. (a) Resonancein a peptide bond (b) ‘Glu–Ser–Val’ tripeptide showingφ, ψ dihedral angles. Carbon(green), nitrogen (blue), andoxygen (red). Inset showsthe repeating backbone inproteins.

During the translation, proteins are synthesised based on the tem-plate of genetic information (RNA) on the ribosome, an intra-cellular organelle, and are spontaneously folded in situ into theirthree-dimensional (3D) structures.

3. Peptide Bond and Dihedral Angles

LinusLinus Pauling andRobert Corey deduced

the crystal structures ofamino acids, peptides,

and other simplerfragments related to

proteins to obtain theinter-atomic distances,bond angles, and other

configurationalparameters that would

allow the reliableprediction of protein

structure.

Pauling and Robert Corey deduced the crystal structures ofamino acids, peptides, and other simpler fragments related to pro-teins to obtain the inter-atomic distances, bond angles, and otherconfigurational parameters that would allow the reliable predic-tion of protein structure [1]. These structural chemists adopted abottom-up approach to build the structure of a protein from theirsmaller constituents. Pauling had predicted partial (40%) dou-ble bond character of a peptide bond because of the resonance ofelectrons caused by the difference in electronegativities (polarity)between the carbon and oxygen atoms in carbonyl group, and thepresence of lone pair of electrons on the adjacent nitrogen along

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the polypeptide main chain [1] (Figure 1a). As a consequence, thepeptide bond is planar and adopts only the trans configuration; acis configuration is allowed for proline, a cyclic amino acid. Forevery amino acid in a polypeptide chain, two single bonds bearingthe torsion angles between N–Cα(φ) and Cα–C(ψ) are rotatableand are primarily responsible for the conformational flexibility ofthe protein structure (Figure 1b).

4. Protein Structure Hierarchy and the Ramachandran Plot

Protein structure hierarchy comprises four levels – primary, sec-ondary, tertiary and quaternary. The linear amino acid sequenceof a protein is defined as its ‘primary structure’. Two main struc-tural features (later referred to as protein secondary structuralelements) in proteins were proposed by Pauling, Corey andHerman Branson [2] based on the hydrogen bonding interac-tions of the protein main chain (backbone). In a right-handedα-helix, the main chain spirals in a right-handed conforma-tion, the oxygen of C=O forms a hydrogen bond with the N–Hlocated four residues away and the side chains are perpendicular

Figure 2. (a) Peptide inα-helical conformation (b)Peptide in β-sheet confor-mation. Dashed lines indi-cate hydrogen bonds.


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to the helical axis (Figure 2a). The stretching of α-helix formsan extended structure referred to as the β-strand. A β-strand isthe planar arrangement of a polypeptide chain with side chainsprojected alternatively on both sides. These strands align to formβ-sheets (anti-parallel and parallel) stabilised by hydrogen bondsbetween the backbone atoms (Figure 2b).

TheG N Ramachandran andco-workers constructedhard sphere models of

the available crystalstructures to evaluate the

extent of rotation aboutthe single bonds alongthe peptide backbone

that can avoid any stericclashes with the atoms in

the vicinity.

Indian biophysicist G N Ramachandran and co-workers (1963)[3] from the University of Madras, India, constructed hard spheremodels of the available crystal structures to evaluate the extent ofrotation about the single bonds along the peptide backbone thatcan avoid any steric clashes with the atoms in the vicinity. Theparametrization of the protein backbone for φ and ψ (torsion an-gles) in proteins was introduced from these studies. Based on theperiodicity of the φ and ψ values, secondary structural elements inproteins (α-helices and β-sheets) are formed, and these two val-ues largely describe the ‘secondary structure’ of a protein. A two-dimensional plot of the sterically allowed φ and ψ values in theprotein backbone was constructed by Ramachandran (Figure 3a).The assessment of the health check of protein structure based onits stereochemical properties is quantified using Ramachandranplot even today. The φ, ψ values for various secondary structuralelements are: right-handed α-helix (-57o, -47o), left-handed α-helix (57o, 47o), parallel β-sheet (-119o, 113o), and antiparallelβ-sheet (-139o, 135o). Glycine can be found anywhere in the Ra-machandran plot as it has high conformational flexibility due tothe lack of a bulky side chain. The secondary structural elementsare connected by loop regions, some of which can be classifiedas β-turns and γ-turns. This causes a reversal in the direction ofpolypeptide chain propagation and generates a spatial arrange-ment of atoms in protein referred to as the protein 3D structure.This 3D structure describes all the atomic positions in a proteinand is referred to as its ‘tertiary structure’. Some proteins requirea higher order assembly, with spatial interactions between two ormore homomeric and heteromeric chains, referred to as ‘quater-nary structure’.


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Figure 3. (a) Ramachan-dran plot of a proteinkinase domain (PDB code:3IK3:B) (b) Schematicrepresentation of PDB code:3IK3:B, N-terminal lobecontains β-strands (flatribbon) and C-terminal lobecontains α-helices (spiral).

5. Protein Function in Health and Disease

Protein folding to a stable conformation often occurs alongsideprotein translation within a biologically reasonable timescale. Someproteins are exported to various organelles or at least detachedfrom the ribosome and moved to the cytoplasm before it folds.Protein folding is sometimes assisted by protein chaperones anddisulfide [S–S] bond forming/isomerizing enzymes in vivo. Onlya correctly folded native protein structure would display the bi-ological function. Proteins function as enzymes, hormones, an-tibodies, structural/transport elements and receptors. Many dis-eases such as Alzheimer’s, Parkinsons, cataract, cystic fibrosis,Creutzfeldt–Jakob disease, etc., results from protein misfolding. Protein folding to a

stable conformationoften occurs alongsideprotein translation withina biologically reasonabletimescale.

6. Protein Data Bank

Experimental methods such as X-ray crystallography, nuclearmagnetic resonance (NMR) and cryo-electron microscopy are


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employed to obtain high-resolution structures of proteins. The co-ordinates of the solved protein structures and other macromoleculesare deposited in the Protein Data Bank (PDB) managed by Re-search Collaboratory for Structural Bioinformatics (www.rcsb.org).The 3D structure of a protein is specified by the x, y, z coordinatesof each of its constituent atoms. Figure 3b shows the graphicalview of the atomic structure of a protein. The figure demonstratesthe complex manner in which a protein main chain folds backand forth, and how the side chains are closely packed (with notwo atoms bumping into each other), so as to create a compactstructure suitable for a precise biological function.

7. The Levinthal Paradox

CyrusA protein of 101 aminoacids will have 100

peptide bonds, and eachrotatable bond in a

protein backbone canhave 3 possible states

and, therefore, thepossible conformationalstates in the protein are

3100. Given that theconformational sampling

about each rotatablebond is 1013 per second,theoretically, this protein

should take 3100 × 1013

seconds or 1027 years tosearch through all the

possible conformations.

Levinthal (1969) proposed a paradox for protein folding[4]. According to him, a protein of say 101 amino acids will have100 peptide bonds, and each rotatable bond in a protein backbonecan have 3 possible states and, therefore, the possible conforma-tional states in the protein are 3100. Given that the conformationalsampling about each rotatable bond is 1013 per second, theoreti-cally, this protein should take 3100 × 1013 seconds or 1027 yearsto search through all the possible conformations. In practice, it isseen that proteins fold in a time scale of milliseconds. How doesa protein search through an exponential number of possible struc-tures, and find the unique structure so quickly? Hence, Levinthalhad first put forward the idea that protein folding does not takeplace by a search of entire conformational space, but it passesthrough some characteristic pathways that facilitate the proteinsto fold into the correct 3D structure.

8. Christian Anfinsen’s Experiments on Protein Folding

The thermodynamic hypothesis of protein folding (Christian BAnfinsen 1972) [5] revealed that the tertiary structure informationof the protein resided in the chemistry of its amino acid sequenceitself. He experimented using ribonuclease A (RNase A) as themodel enzyme; its structure stabilised by four disulfide bonds.


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The protein was treated with urea (protein denaturant to disruptthe hydrogen bonds) and β-mercaptoethanol (reduce the disulfidebonds). Removal of the reducing agent in the presence of urearegained only 1% of the protein activity in accordance with thefraction of RNase A that would form the correct S–S connectiv-ity. However, when both urea and β-mercaptoethanol were re-moved from the denatured RNase A, it folded back to the activeand functional form.

9. Protein Folding and Architecture

For a specific environmental condition of a protein such as sol-vent, the fundamental principles of protein folding are universal.Given Given that the linear

structure of a protein hasa highly flexiblebackbone, and hencethere are severaltheoreticalconformationalpossibilities, it isimportant to understandthe factors that governthe folding of an aminoacid sequence into a 3Dprotein structure.

that the linear structure of a protein has a highly flexiblebackbone, and hence there are several theoretical conformationalpossibilities, it is important to understand the factors that governthe folding of an amino acid sequence into a 3D protein structure.The simultaneous presence of several non-bonding interactionsis observed in a protein’s 3D structure. Hydrophobic forces, hy-drogen bonding, electrostatic and van der Waals forces of attrac-tions dictate the intramolecular interactions. The unfolded pro-tein in water (presence of 70% water in a living cell) is highlysolvated and stabilised by all possible hydrogen bonding inter-actions. However, the polypeptide chain in water (solvent) envi-ronment leads to the segregation of hydrophobic and hydrophilicresidues. The non-polar residues interact with each other to forma hydrophobic core. It is widely observed that protein foldingis driven by hydrophobic interactions. It is the preference ofnon-polar residues to reduce contact with polar water and asso-ciate with like non-polar residues. The aggregation of non-polargroups in water leads to an increase in entropy owing to the re-lease of water molecules into bulk water. The core of the proteinis hydrophobic and tightly packed with a packing density (pack-ing fraction) of 0.74 [6] which is comparable to the packing den-sity of organic molecules (0.7 to 0.78). Packing density is re-ferred to as the ratio of the volume enclosed by the van der Waalssurface of a protein to the actual volume of the occupied space.


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While the core of the protein can be compared to a solid due tothe close atomic packing, the surface residues form the flexibleregions such as loops that resemble liquid state. Thus, proteinfolding represents a liquid to solid state transition. However, theformation of the folded state of a protein constrains its conforma-tional flexibility, and this restriction is entropically unfavourable!Electrostatic forces of attraction exist between two atoms bearingopposite charges when they are separated by nearly 3Å distance.These interactions within a protein help stabilize its structure, andthe energy of interaction can be quantified by Coulomb’s law.

AThe cumulativenon-bonding forces and

electrostatic interactionstogether stabilise the

protein structure, and ahighly

thermodynamicallystable structure with

lowest energy (globalminimum) is favoured.

The energy ofinteractions in a protein

can be quantified byCoulomb’s law.

hydrogen bond interaction exists when a hydrogen atom ispartly shared between two relatively electronegative atoms suchas nitrogen, oxygen, or halogen separated by 2.5 to 3.2 Å dis-tance. A hydrogen bond donor refers to a group where the hydro-gen is covalently bonded to an electronegative atom, and the elec-tron density is pulled away from the hydrogen creating a partialpositive charge (δ+). The hydrogen devoid of the electron cloudinteracts with another electronegative atom which is a hydrogenbond acceptor. An essential criterion for a hydrogen bond isthat both electronegative atoms that share the hydrogen should benearly collinear. The electron charge distribution around an atommay not be uniform throughout and depends on the influence ofneighbouring atoms to induce a dipole. The van der Waals forcesof attractions on closely packed atoms are influenced by induceddipole-dipole attractions and add to the structural stability of theproteins. The typical bond energies of various interactions are:ionic (4–15 kcal mol−1), hydrogen bonding (2–10 kcal mol−1),van der Waals (0.5–1.0 kcal mol−1), and hydrophobic (1.3 kcalmol−1). For the sake of comparison, note that the bond energyof a typical carbon-carbon (C–C) covalent bond is 85 kcal mol−1.These cumulative non-bonding forces together stabilise the pro-tein structure, and a highly thermodynamically stable structurewith lowest energy (global minimum) is favoured.

The solvent water molecules heavily solvate the solute proteinson the exterior and to some extent restrict the flexibility of sidechains on the protein surface. However, these water molecules


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are in constant exchange with one another and form a water layeron the outside of protein structure (the catalytic waters and thosein the interior of the protein structure are less mobile). The in-tramolecular interactions in proteins are often stabilised by theformation of covalent (S–S) bond between the thiol groups of twocysteine residues. The buried core of the protein has hydropho-bic interactions or hydrogen bond and ion pair interactions. If acharged side chain of a protein is located in the protein interior, itmay be required for the catalytic activity. The side chain confor-mations of amino acids are dependent on the backbone confor-mations, and this information is available in side chain rotamerlibraries [7]. Thermodynamics The correctly folded

protein 3D structure isstable compared to thelinear chain by only12–40 kcal mol−1.

involves the measurement of pro-tein stability which is the difference in free energy between un-folded and folded (native) states. The correctly folded protein 3Dstructure is stable compared to the linear chain by only 12–40 kcalmol−1. The protein structures can be classified into all-α (proteinmainly with α -helices), all-β (protein mainly with β -strands), α/β (protein chain alternates between α-helices and β-strands) andα + β proteins (α-helices and β-strands are segregated in distinctlocations in the protein structure) (Figure 4).

10. Protein Dynamics and Functions

The correctly folded 3D structure of a protein creates a cavity(or cleft) for the binding of a substrate/cofactor in its active site.When the structures of a protein in the apo-form (protein only)and holo-form (bound to ligand) are compared, certain confor-mational changes in the ligand binding region are observed. This‘induced fit model’ was proposed by Daniel Koshland (1963) [8].According to the induced fit model, the binding of a ligand to theprotein active site brings about a conformational change in the lig-and as well the conformation of active site residues in the proteinpocket, such that the precise recognition between protein-ligandcomplex permits the enzyme catalysed reactions to proceed at sig-nificantly higher rates than uncatalysed reactions [9].

In some cases, allostery plays a crucial role in protein function.


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Figure 4. Schematic repre-sentation of (a) all-α, (b) all-β (c) α/β (d) α + β proteins.

Ligand binding to one region of a protein causes a conformationalchange elsewhere, thereby opening up or unravelling a new bind-ing site that was unavailable earlier. Some proteins called ‘in-trinsically disordered proteins’ possess only partial sub-structuresand lack a fixed or ordered 3D structure. Such proteins gain their3D structure and function in suitable environmental conditionsor in the presence of their folding partners mediated via protein-protein interactions.

ProteinProtein structures aredynamic and alternate

between active andinactive forms throughsubtle conformational

changes. These motionsin protein structures can

be studied usingmolecular dynamics

simulations.

structures are dynamic and alternate between active andinactive forms through subtle conformational changes. Thesemotions in protein structures can be studied using molecular dy-namics simulations. Certain protein structures are stabilised andrendered functionally active in the presence of organic and in-organic cofactors. For example, haemoglobin, an oxygen trans-porter, is a tetrameric (two α and two β subunits) protein andbinds haem cofactor and oxygen. The oxyhaemoglobin (oxy-


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gen bound) and deoxyhaemoglobin vary structurally, and a sin-gle amino acid residue alteration in haemoglobin can lead to dis-eases. For instance, the mutation of Glu to Val (structures of Gluand Val can be seen in Figure 1b) at sixth position in the β-chainof haemoglobin causes sickle cell disease.

11. Protein Domains

Some Domain organization inproteins areadvantageous to folding,function and structuralstability, and hence theycan be inserted anywherealong the proteinsequence.

small proteins of length 50–200 amino acids often fold intoa single domain protein, and large proteins fold into multi-domainproteins, with each domain performing a distinct function. Adomain is an individual structural (folding) and functional unitindependent of the rest of the protein. Domain organization inproteins are advantageous to folding, function and structural sta-bility, and hence they can be inserted anywhere along the proteinsequence. These domains are connected by short peptides to en-able them to adopt a variety of conformations that are essentialfor protein-protein interactions and in the regulation of allostericnature of proteins.

12. Conclusion

Proteins are composed of amino acids. The amino acid sequencedictates the 3D structure of the protein. Several non-bonded,short, medium and long-range interactions mediate protein fold-ing to a thermodynamically stable structure. Protein structuresare mainly classified as all-α, all-β, α/β or α + β. X-ray crys-tallography, NMR and cryo-electron microscopy are experimen-tal techniques used for protein structure determination. A cor-rectly folded protein structure elicits the biological function, andits misfolding is associated with various disorders. The flexibilityof protein structure allows it to modulate its functional role andmediate interactions with other binding partners.

Acknowledgements

I thank School of Chemistry, University of Hyderabad for the re-search facilities and my students for enabling a continuous learn-


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ing process.

Suggested Reading

[1] L Pauling, R B Corey, Configurations of Polypeptide Chains With FavoredOrientations Around Single Bonds. Two New Pleated Sheets, Proc. Natl. Acad.Sci. USA, Vol.37, No.11, pp.729–740, 1951.

[2] L Pauling, R B Corey, H R Branson, The Structure of Proteins: Two Hydrogen-bonded Helical Configurations of the Polypeptide Chain, Proc. Natl. Acad. Sci.USA, Vol.37 No.4, pp.205–211, 1951.

[3] G N Ramachandran, C Ramakrishnan, V Sasisekharan, Stereochemistry ofPolypeptide Chain Configurations, J. Mol. Biol., Vol.7, pp.95–99, 1963.

[4] C Levinthal, How to Fold Graciously, Mossbaun Spectroscopy in Biological Sys-tems Proceedings, Univ. of Illinois Bulletin, Vol.67, No.41, pp.22–24, 1969.

[5] C B Anfinsen, Principles that Govern the Folding of Protein Chains, Science,Vol.181, No.4096, pp.223–230, 1973.

Address for Correspondence

Lalitha Guruprasad

School of Chemistry

University of Hyderabad

Hyderabad 500 046, India.

Email:

lalitha.guruprasad@uohyd.

ac.in

[6] F M Richards, The Interpretation of Protein Structures: Total Volume, GroupVolume Distributions and Packing Density, J. of Mol. Biol., Vol.82, No.1, pp.1–14, 1974.

[7] R Dunbrack Jr, M Karplus, Conformational Analysis of the Backbone-Dependent Rotamer Preferences of Protein Sidechains, Natu. Struc. Biol.,Vol.1, No.5, pp.334–340, 1994.

[8] D E Koshland Jr, Correlation of Structure and Function in Enzymatic Reac-tion, Science, Vol.142, No. 3599, pp.1533–1541, 1963.

[9] Jeremy M Berg, Lubert Stryer, John L Tymoczko, Gregory J Gatto, Biochem-istry, (8th Edition), Publishers: WH Freeman, April 2015.


GENERAL ARTICLE Protein Structure - ias

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