Three-Dimensional Structure of Protein

• Proteins that consist of a single polypeptide chain are generally considered at three levels of organization: primary, secondary, and tertiary structure. 
• For proteins that contain two or more polypeptide chains, each chain is a subunit and there is a quaternary level of structure. 
• The primary structure is that the distinctive sequence of amino acids that frame a specific polypeptide; covalent bonds maintain primary structure; secondary, tertiary, and quaternary structures are maintained mainly by noncovalent bonds and disulfide bridges may also be considered at the secondary and tertiary levels. 
• Secondary structure arises from repeated hydrogen bonding within a chain, as in the α-helix, β-pleated sheet, and β-turn. 
• Tertiary structure describes the three-dimensional stereochemical relationships of all of the amino acid residues in a single protein chain. 
• Folding of a polypeptide is an orderly sequential process by which the polypeptide attains the lowest possible state of energy. 
• The polypeptide folding into its secondary structure is decided mainly by the primary structure. 
• Once the secondary structures are in place, a tertiary structure is formed and stabilized by interactions among amino acids which may be far from each other in the primary sequence but which are close to each other in the three-dimensional structure. 
• Proteins depend upon the stable conformations for their specific biological functions. 
• A functional protein which is more stable will be in its native form. 
• The three dimensional conformation of a polypeptide chain is finally determined by its amino acid sequence. 
• Changes in that sequence, as they arise from mutations in DNA, may yield conformationally altered proteins. Since the biological function of a protein depends on a particular conformation, changes such as denaturation can lead to loss of biological activity.

Attractive and Repulsive Forces:

Both attractive and repulsive interactions occur among different regions of polypeptide chains and are responsible for most secondary and tertiary structure.

Attractive Forces:

Covalent Bonds:

Covalent bonds involve the equal sharing of an electron pair by two atoms. Examples of covalent bonds are peptide and disulfide bonds between amino acids, and C-N, C-O, and C-C bonds within amino acids.

Coordinate Covalent Bonds: 

Coordinate covalent bonds involve the unequal sharing of an electron pair by two atoms, with both electrons coming from the same atom. The electron pair donor is the ligand, or Lewis base, whereas the acceptor is the central atom or Lewis acid. These bonds are important in all interactions between transition metals and organic ligands like Fe2+ in hemoglobin and the cytochromes.

Ionic Interactions:

Ionic interactions arise from electrostatic attraction between two groups of opposite charge. These bonds are formed between positively charged side chains and negatively charged groups. 

Hydrogen Bonds:

Hydrogen bonds involve the sharing of a hydrogen atom between two electronegative atoms that have unbonded electrons. These bonds, although weaker bonds than above, are important in water-water interactions. In proteins, groups possessing a hydrogen atom that can be shared include N-H (peptide nitrogen, imidazole, and indole),-SH (cysteine),-OH (serine, threonine, tyrosine, and hydroxyproline),-NH2 and-NH3+- (arginine, lysine, and ε-amino), and-CONH2 (carbamino, asparagine, and glutamine). Groups capable of sharing a hydrogen atom include - COO - (aspartate, glutamate, and α-carboxylate),-SCH3 (methionine),-S-S-(disulfide), and -C=O (in peptide and ester linkages.

Van der Waals Attractive Forces:

Van der Waals attractive forces are due to a fixed dipole in one molecule that induces rapidly oscillating dipoles in another molecule through distortion of the electron cloud. Dipole's positive end will attract an electron cloud toward it; the negative end will repel it away. The strength of these interactions is mainly dependent on distance, varying as 1/r 6 where r is the inter atomic separation. The Van der Waals forces are particularly important in the nonpolar interior structure of proteins, where they provide attractive forces between nonpolar side chains. Hydrophobic interactions cause nonpolar side chains to cling together in polar solvents, especially water. These interactions do not produce true "bonds," since there is no sharing of electrons between the groups involved. The groups are pushed together by their "expulsion" from the polar medium. Such forces are also involved  in lipid-lipid interactions occur in membranes.

Repulsive Forces:

• Electrostatic repulsion occurs between charged groups of the same charge and is the opposite of ionic forces. This kind of repulsion acts according to Coulomb's law: ql q2/r 2, where ql and q2 are the charges and r is the interatomic separation.
• Van der Waals repulsiveforces operate between atoms at very short distances from each other and result from the dipoles induced by the mutual repulsion of electron clouds. Since there is no involvement of a fixed dipole (in contrast to van der Waals attractive forces), the dependence on distance in this case is even greater (1/rl2). 
• These repulsive forces operate when atoms not bonded to each other approach more closely than the sum of their atomic radii and are the underlying forces in steric hindrance between atoms.

Primary Structure:

Peptide Bond:

• Peptide bonds have a planar trans configuration and undergo very little rotation or twisting around the amide bond that links the ε-amino nitrogen of one amino acid to the carbonyl carbon of the next. 
• This effect is due to amido-imido tautomerization. 
• Electrons are shared by the nitrogen and oxygen atoms, and the N-C and C-O bonds are both "one-and-one-half" bonds. 
• The short carbonyl carbon-nitrogen bond length, 0.132 nm whereas the usual carbon-nitrogen single bond length is 0.147 nm. 
• The peptide bond's planarity and rigidity are accounted for by the truth that free rotation cannot occur around double bonds. 
• Whereas most peptide bonds exist in the trans configuration to keep the side chains apart, the peptide bond that involves the - NH group of the rigid pyrrolidone ring of proline can occur in both trans and cis arrangements. 
• However, x-ray data suggest that the trans form occurs more frequently in proteins than does the cis form. It has been further postulated that some proline residues can exist in either the cis or trans configuration. 
• The bonds on either side of the α-carbon are strictly single bonds. Rotation is possible around single bonds. 
• The angle of rotation of the α-carbon-nitrogen bond is designated by φ. α carbon-carbonyl carbon bond is designated by ψ. A polypeptide has specific φ and ψ values for each residue that determines its conformation.

Peptide bond and it's geomety

The linear sequence of a protein is mainly considered as Primary structure. The primary structure of a protein can be mentioned beginning from the amino-terminal (N) end to the carboxyl-terminal (C) end.

Primary Structure

Secondary Structure:

• The folding of polypeptide chains into ordered structures maintained by repetitive hydrogen bonding is called secondary structure. 
• Linus Pauling and Robert Corey experiments explained the secondary structures of protein
• The most common types of secondary structure are the right-handed α-helix, parallel and antiparallel β-pleated sheets, and β-turns. 
• The absence of repetitive hydrogen-bonded regions may also be part of secondary structure. α -keratin of hair and fibroin of silk contain mostly α-helix and β-pleated sheet, respectively. 
• Hemoglobin has both or-helical and non-hydrogen-bonded region. Globularproteins usually have mixed and fibrous proteins have predominantly one kind of secondary structure.

α-Helix:

• The rod-shaped right-handed α-helix, one of the most common secondary structures found in naturally occurring proteins.
• In the right-handed α -helix the helix turns counterclockwise (C-terminal to N-terminal) and in the left-handed it turns clockwise. 
• The left-handed α-helix is less stable than a right-handed α -helix because its carbonyl groups and the R-groups are sterically hindered. 
• The helical structure is stabilized by intrachain hydrogen bonds involving each -NH and-CO group of every peptide bond. 
• These hydrogen bonds are parallel to the axis of the helix and form between the first residue and the fourth residue, and so on, producing 3.6 amino acid residues per turn of the helix. 
• The rise per residue is 0.15 nm and the one turn's length is 0.54 nm. In some proteins like α-keratin, myoglobin, and hemoglobin, α-helices contribute significantly to the secondary structure. 
• Destabilization of an α-helix may occur for a variety of reasons: electrostatic repulsion between similarly charged R-groups (Asp, Glu, His, Lys, Arg); steric interactions due to bulky substitutions on the β-carbons of neighboring residues (Ile, Thr); and formation of side-chain hydrogen or ionic bonds. 
• Glycine residues can be arranged in an α -helix; however, the preferred and more stable conformation for a glycine-rich polypeptide is the β-pleated sheet because the R-group of glycine (-H) is small and gives rise to a large degree of rotational freedom around the -carbon of this amino acid. 
• Prolyl and hydroxyprolyl residues usually create a bend in an a-helix because their α-nitrogen atoms are located in rigid ring structures that cannot accommodate the helical bonding angles. 
• Moreover, they do not have an amido hydrogen and therefore can form neither the necessary hydrogen bond nor the usual planar peptide bond. 
• However, some proteins such as rhodopsin do contain proline residues embedded in α-helical segments. 
• In some proteins, the α-helices twist around each other to form rope-like structures to give rise to a supersecondary structure. 
• Examples of such proteins are the α-keratins, which are major protein components of hair, skin, and nails. 
• These proteins are rich in amino acid residues that favor the formation of an α-helix. 
• In addition, consistent with their properties of water insolubility and cohesive strength, α-keratins are rich in hydrophobic amino acid residues and disulfide cross-links. 
• The α-helices are arranged parallel to their length with all the N-terminal residues present at the same end. Three α-helical polypeptides are intertwined to form a left handed supercoil, called a protofibril. 
• Eleven protofibrils form a microfibril. The polypeptides within the supercoil are linked together by disulfide linkages and are also stabilized by van der Waals forces between the nonpolar side chains. 
• The number of disulfide cross-linkages in α-keratins varies from one source to another. Skin is stretchable because of fewer cross-links, whereas nails are inflexible and tough because of many more cross-links.

α-Helix

β-Pleated Sheet:

• The β -structure has the amino acids in an extended confirmation with a distance between adjacent residues of 0.35 nm. 
• The structure is stabilized by intermolecular hydrogen bonds between the - NH and - CO groups of adjacent polypeptide chains. 
• The β-structure can occur between separate peptide chains or between segments of the same peptide chain, where it folds back upon itself. 
• Two types of β-pleated sheets exist: parallel and antiparallel. In the parallel sheet structure, adjacent chains are aligned in the same direction with respect to N-terminal and C-terminal residues, whereas in the antiparallel sheet structure, the alignments are in the opposite directions. 
• Silk fibroin consists almost entirely of antiparallel β-structures, every other amino acid is glycine and alanine predominates in the remaining positions. 
• The β-pleated sheet occurs as a principal secondary structure in proteins found in persons with amyloidosis. The proteins that accumulate are called amyloid and are aggregates of twisted β-pleated sheet fibrils. 
• They derive from endogenous proteins on selective proteolysis and other chemical modifications. The fibrillar proteins are insoluble and relatively inert to proteolysis. 
• Their accumulation in tissues and organs can severely disrupt normal physiological processes. 
• The amyloid deposit, which occurs in several different tissues, is produced in certain chronic inflammatory diseases, in some cancers, and in the brain with some disorders, e.g., Alzheimer's disease. Partial or total disappearance of amyloid deposits in mice has been noted on administration of dimethyl sulfoxide, which disrupts hydrogen bonds.

β-Pleated Sheets

β-Turns:

β-Turns which are stabilized by a hydrogen bond, cause polypeptide chains to be compact molecules. The four amino acid residues of a β-turn form a hairpin structure in a polypeptide chain, thus providing an energetically economical and space-saving method of turning a corner. Two tetrapeptide conformations can accomplish a β-turn that is stabilized by a hydrogen bond.

β-turns

Random Coil:

• Certain regions of peptides may not possess any definable repeat pattern in which each residue of the peptide chain interacts with other residues, as in an α -helix. However, a given amino acid sequence has only one conformation, or possibly a few, into which it coils itself. 
• This conformation has minimal energy. Since energy is required to bring about change in protein conformation, the molecule may remain trapped in a conformation corresponding to minimal energy, even though it is not at absolute minimum internal energy. 
• This concept of a molecule seeking a preferred, low-energy state is the basis for the tenet that the primary amino acid sequence of proteins determines the secondary, tertiary, and quaternary structures.

Other Types of Secondary Structure:

Other types of protein secondary structure include the type present in collagen, the most abundant of all human proteins. Collagen peptide chains are twisted together into a three stranded helix. The resultant "three-stranded rope" is then twisted into a superhelix.

Tertiary Structure:

Three-dimensional tertiary structure in proteins is maintained by ionic bonds, hydrogen bonds, -S-S - bridges, van der Waals forces, and hydrophobic interactions. The first protein whose tertiary structure was determined is myoglobin, an oxygen-binding protein consisting of 153 amino acid residues. Its structure was deduced from x-ray studies by Kendrew, Perutz, et al. after a 19-year analysis. Their studies provided not only the first three-dimensional representation of a globular protein but also insight into the important bonding modes in tertiary structures. The major features of myoglobin are described below:
1. Myoglobin is an extremely compact molecule with very little empty space, accommodating only a small number of water molecules within the overall molecular dimensions of 4.5 x 3.5 x 2.5 nm. All of the peptide bonds are in trans configurations to each other.
2. Eight right-handed α-helical segments involve approximately 75 % of the chain. Five non helical
regions separate the helical segments. There are two non helical regions, one at the N terminus and another at the C terminus.
3. Eight terminations of the α-helices occur in the molecule, four at the four prolyl residues and the rest at residues of isoleucine and serine.
4. Except for two histidyl residues, the interior of the molecule contains almost all non polar amino acids. The exterior of the molecule contains polar residues that are hydrated and non polar amino acid residues. The amino acid residues whose R-groups contain both polar and non polar portions are oriented so that the nonpolar group faces inward and the polar group faces outward, allowing only the polar portion to come in contact with water.
5. Myoglobin has no S-S bridges that generally help stabilize the conformation formed by amino acid
interactions.

           Triose phosphate isomerase, a glycolytic enzyme, contains a core of eight β-pleated sheets surrounded by eight α-helices arranged in a symmetrical cylindrical barrel structure known as αβ-barrels.

Tertiary Structure

Quaternary Structure:

• Quaternary structure exists in proteins consisting of two or more polypeptide chains. 
• These proteins are called oligomers. 
• The quaternary structure describes the pattern in which subunits are arranged in the native protein. 
• Subunits are joined together by noncovalent interactions; as a result, oligomeric proteins can undergo frequent conformational changes that affect biological activity. 
• Oligomeric proteins include the hemoglobins, allosteric enzymes responsible for the regulation of metabolism, and contractile proteins such as actin and tubulin.

Quaternary Structure

                                               

Denaturation:

Denaturation of a native protein can be explained as a change in its physical, chemical, or biological properties. Mild denaturation might disrupt tertiary or quaternary structures, whereas harsher conditions might fragment the chain. Mild denaturation normally is a reversible process. Some of the changes in properties that may be caused by denaturation are as follows:

1. Decreased solubility.

2. Alteration in the internal structure and arrangement of peptide chains that does not involve breaking the peptide bonds.

3. Disrupted secondary structure.

4. Increased chemical reactivity of functional groups of amino acids, particularly ionizable and sulfhydryl groups.

5. Increased susceptibility to hydrolysis by proteolytic enzymes.

6. Decrease or total loss of the original biological activity.

7. Loss of crystallizability.

                 Studies by Anfinsen of the reversible denaturation of the pancreatic enzyme ribonuclease explained the hypothesis that secondary and tertiary structures are derived inclusively from the primary structure of a protein. RNase A consists of a single polypeptide chain of 124 amino acid residues with four disulfide bonds. Treatment of the enzyme with 8 M urea, which disrupts noncovalent bonds, and β-mercaptoethanol, which reduces disulfide linkages to cysteinyl residues, yields a random coil conformation. However, if both reagents are removed and the cysteinyl residues are allowed to oxidize and re-form disulfide bonds, of the 105 different possible intramolecular combinations of disulfide linkages, only the four correct bonds form, and the denatured enzyme renatures to its original, biologically active structure. These experiments are taken as proof that the primary structure determines the unique three-dimensional structure of a protein.

Denaturation of RNase A