1.7 CONFORMATION OF PROTEINS

Amino Acids

 Amino acids are the building blocks of protein.  The primary functions of amino acids are to build muscle tissue.  More than 300 amino acids exist in nature.  Only 20 aa are used by the living organisms.  10 Amino acids can be synthesized by the body itself.  All 20 of the common amino acids are α amino acids.

Structure of Amino acid

Amino acids differ from each other in their side chains/R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water. All the common amino acids except glycine, the carbon is bonded to four different groups: 1. a carboxyl group 2. an amino group 3. an R group 4. a hydrogen atom The -carbon atom is thus a chiral center. So they are optically active. In glycine, the R group is another hydrogen, so optically inactive They form enantiomers. The Amino Acid residues in proteins are L-stereoisomers.

Figure 1.7.1 General structure of an amino acid. This structure is common to all but one of the -amino acids. (Proline, a cyclic amino acid, is the exception.) The R group or side chain attached to the α carbon is different in each amino acid.

Figure 1.7.2 Stereoisomerism in α-amino acids.

Classification of Amino Acids They are classified based on the properties of their R groups

 Polar amino acids –Serine, Threonine, Glutamine, Cysteine, Asparagine  Non-polar amino acids –Glycine, Valine, Alanine, proline, Leucine, Isoleucine, Methionine  Amino acids with negatively charged R group– Aspartic Acid, Glutamic acid.  Amino acids with Positively charged R group- Lysine, Arginine, Histidine  Amino acids with Aromatic R group- Phenyalanine, Tyrosine,

Classification of Amino Acids -Based on functional group

Polar

Positively charged Uncharged Negatively charged

 Aliphatic: gly (G), ala (A) , val (V), leu (L), ile (I)  Aromatic: Trp (W), Phe (F), Tyr (Y), His (H),

Non Polar

Aliphatic Aromatic

Unit 1  Sulphur : Met (M), Cys (C)  Hydroxyl: Ser (S), Thr (T), Tyr (Y)  Cyclic: pro (P)

 Carboxyl: asp (D), glu (E)  Amine: lys (K), arg (R)

Figure 1.7.3 The 20 common amino acids of proteins. The structural f ormulas show the state of ionization that would predominate at pH 7.0.

Uncommon Amino Acids

 4-hydroxyproline- plant cell wall proteins, collagen  5-hydroxylysine- collagen  6-NMethyllysine- constituent of myosin  Carboxyglutamate- bloodclotting protein prothrombin and Ca2+ binding proteins  Desmosine - a derivative of four Lys - Elastin.  Selenocysteine is introduced during protein synthesis rather than created through a postsynthetic modification. It contains selenium rather than the sulfur of cys. It is present at the catalytic site of  glutathione peroxidase  formate dehydrogenase  Azaserine – antibiotic  Pyrolysine- bacterial proteins  Ornithine &Citrulline - Key intermediates in biosynthesis of Arginine and urea cycle.

Amino Acids Can Act as Acids and Bases

 When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion. o A zwitterion can act as either an acid (proton donor) or a base (proton acceptor)  Substances having this dual nature are amphoteric and are often called ampholytes (from “amphoteric electrolytes”).  pI of Amino Acids  Amphoteric molecules called zwitterions. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+).  The pI is the pH value at which the molecule carries no electrical charge or the negative and positive charges are equal.  At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge.  Amino acids are ampholytes, pKa of α-COOH is =2 and of α-NH2 is = 9  The COOH group loses the proton first when compared to NH3 group.

Peptide bond Formation

 Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide.  It is formed by removal of water (dehydration) from the α-carboxyl group of one amino acid and the α–amino group of another.  It is a type of condensation reaction.  It is an Endergonic reaction with ΔG=+21KJ/mol.

Figure 1.7.4 Tit ration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25 C. The ionic species predominating at key points in the titration are shown above the graph.

Properties of Peptide Bond

 Planar (2 α-C & -O=C-N-H in one plane).  Partial double bond character due to resonance structures of peptide bond (bond length is 1.32 A).  Trans configuration due to steric hindrance.

Figure 1.7.5 Titration curves f or (a) glutamate and (b) histidine.  Freedom of rotation The 2 bonds around the α-carbon have freedom of rotation making proteins flexible to bend and fold.

Peptide Bond Angles

 Proposed by Linus Pauling and Robert Corey o The α carbons of adjacent amino acid residues are separated by three covalent bonds  Findings o X-ray diffraction studies of crystals of amino acids and of simple dipeptides and tripeptides demonstrated that o peptide C--N bond is somewhat shorter than the C--N bond in a simple amine and that the atoms associated with the peptide bond are coplanar. o This indicated a resonance or partial Figure 1.7.6 Formation of a peptide bond by condensation. sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen.

Why Partial Double bond?

 The oxygen has a partial negative charge and the nitrogen a partial positive charge, setting up a small electric dipole.  The 6 atoms of the peptide group lie in a single plane, with the oxygen atom of the carbonyl group and the hydrogen atom of the amide nitrogen trans to each other.

Unit 1  These findings Pauling and Corey concluded that the peptide C--N bonds are unable to rotate freely because of their partial double-bond character. Rotation is permitted about the N-- Cα and the C-Cα bonds.

Bond Denotation

 ω = C-N  Ѱ = Cα-C  Φ = N-Cα  K = Cα-R

Proteins

 Proteins play crucial functional roles in all biological processes:  enzymatic catalysis  signaling messengers  structural elements  Function depends on unique 3-D structure.

Protein Conformation

The spatial arrangement of atoms in a protein is called its conformation. The possible conformations of a protein include any structural state that can be achieved without breaking covalent bonds. A change in conformation could occur by rotation about single bonds. Of the numerous conformations that are theoretically possible in a protein containing hundreds of single bonds, a few generally predominate under biological conditions. The need for multiple stable conformations reflects the changes that must occur in most proteins as they bind to other molecules or catalyze reactions. The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable, having the lowest Gibbs free energy (G).  Proteins in any of their functional, folded conformations are called native proteins.  A protein’s conformation is stabilized largely by weak interactions o Hydrogen bonds o Ionic interactions o Hydrophobic interactions

Figure 1.7.7 Rotation of Bonds around the peptide .

Ramachandran Plot

 Allowed values for Ф and ψ are graphically revealed when Ф versus ψ is plotted in a Ramachandran plot, introduced by G. N. Ramachandran  Bond Angles For Different Proteins

Structure of Proteins

 Primary structure: aa sequence  Secondary structure: regular chain organization pattern  Tertiary structure: 3D complex folding  Quarternary structure: association between polypeptides.

Secondary Structures

Figure 1.7.8 The structural hierarchy in proteins. (a) Primary structure, (b) secondary structure, (c) tertiary structure, and (d) quaternary structure.

 Secondary structure is the initial folding pattern (periodic repeats) of the linear polypeptide  3 main types of secondary structure: o α- helix, o β-sheet and o Β- bend/loop  Secondary structures are stabilized by hydrogen bonds.

α-helix

 It is right-handed or clock-wise left-handed helix is not viable due to steric hindrance  Each turn has 3.6 aa residues and is 5.4 A high

Figure 1.7.9 The Ramachandran diagram. It shows the sterically allowed and angles f or poly-L-alanine and was calculated using the van der Waals distances in Table 8-1. Regions of “normally allowed” and angles are shaded in blue, whereas greenshaded regions correspond to conformations having “outer limit” van der Waals distances.The conformation angles,  and , of several secondary structures are indicated below:

 The helix is stabilized by H-bonds between –N-H and –C=O groups of every 4th amino acid  The amino acid residues in the helix have conformations with ψ=-45 to 50 and Ф=- 60

Role of Amino Acids in Alpha Helix formation

Five different kinds of constraints affect the stability of an α helix: o the electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups o the bulkiness of adjacent R groups o the interactions between R groups spaced 3/4 residues apart o the

ccurrence of Pro and Gly residues o the interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the helix  long block of Glu residues – cannot form α helix – negatively charged carboxyl groups of adjacent

Glu residues repel each other  long block of Lys and/or Arg residues- cannot form α helix positively charged carboxyl groups of adjacent residues repel each other  The bulk and shape of Asn, Ser, Thr, and Cys - can also destabilize an α helix if they are close together in the chain.

Parallel beta sheets

Antiparallel beta sheets

Figure 1.7.10 Parallel and Anti-parallel beta Sheets; The Parallel Beta-Sheet is characterized by two peptide strands running in the same direction held together by hydrogen bonding between the strands; The Antiparallel Beta-Sheet is characterized by two peptide strands running in opposite directions held together by hydrogen bonding between the strands.

Unit 1  Ion pair- Positively charged amino acids are often found three residues away from negatively charged amino acids, permitting the formation of an  Two aromatic amino acid residues are often similarly spaced, resulting in a hydrophobic interaction  Pro residue introduces a destabilizing kink in an α helix  Glycine occurs infrequently in α helices as it has more conformational flexibility than the other amino acid residues.  The four amino acid residues at each end of the helix do not participate fully in the helix hydrogen bonds  The partial positive and negative charges of the helix dipole actually reside on the peptide amino and carbonyl groups near the amino-terminal and carboxyl-terminal ends of the helix, respectively.  Negatively charged amino acids are often found near the amino terminus of the helical segment, where they have a stabilizing interaction with the positive charge of the helix dipole; a positively charged amino acid at the amino terminal end is destabilizing.

β-Pleated Sheet

 Extended stretches of 5 or more aa are called β- strands  β-strands organized next to each other make β-sheets  If adjacent strands are oriented in the same direction (N-end to C-end), it is a parallel β-sheet, if adjacent strands run opposite to each other, it is an antiparallel β-sheet. There can also be mixed βsheets  H-bonding pattern varies depending on type of sheet  β-sheets are usually twisted rather than flat  Fatty acid binding proteins are made almost entirely of β-sheets

Bend / Loop

 Polypeptide chains can fold upon themselves forming a bend or a loop.  Usually 4 aa are required to form the turn  H-bond between the 1st and 4th aa in the turn  Bends are usually on the surface of globular proteins  Proline residues frequently found in bends / loops

Tertiary Structure

 3D folding or ‘bundling up’ of the protein.  Non-polar residues are buried inside, polar residues are exposed outwards to aqueous environment

Figure 1.7.11 Formation of Protein tertiary structure. Tertiary structure is formed by the packing of protein secondary structure elements into compact globular units called protein domains. Visit: http://study.biotecnika.org for colored picture.

Unit 1  Many proteins are organized into multiple ‘domains’  Domains are compact globular units that are connected by a flexible segment of the polypeptide  Each domain is contributes a specific function to the overall protein  Different proteins may share similar domain structures, eg: kinase-, cysteine-rich-, globin-domains.

5 kinds of bonds stabilize tertiary structure

1. H-bonds 2. van der waals interactions 3. hydrophobic interactions 4. ionic interactions 5. disulphide linkages

In disulphide linkages, the SH groups of two neighboring cysteines form a –S-S- bond called as a disulphide linkage. It is a covalent bond, but readily cleaved by reducing agents that supply the protons to form the SH groups again. Reducing agents include β-mercaptoethanol α helices and β sheets generally are found in different structural layers. This is because the backbone of a polypeptide segment in the β conformation cannot readily hydrogen-bond to an α helix aligned with it.

Domains

 Polypeptides with more than a few hundred amino acid residues often fold into two or more stable, globular units called domains.  a domain retains its correct three-dimensional structure even when it is separated (for example, by proteolytic cleavage) from the remainder of the polypeptide chain.  A protein with multiple domains may appear to have a distinct globular lobe for each domain, but, more commonly, extensive contacts between domains make individual domains hard to discern.  Different domains often have distinct functions, such as the binding of small molecules or interaction with other proteins. Small proteins usually have only one domain (the domain is the protein).

Supersecondary Structure/ Motifs/ Folds

 Are stable arrangements of several elements of secondary structure and the connections between them  range from simple to complex, sometimes appearing in repeating units or combinations  A single large motif may comprise the entire protein e.g. the coiled coil of α-keratin  formed to bury hydrophobic amino acid R groups so as to exclude water [requires at least two layers of secondary structure].

Classification

 Simple Types- o β-α-β loop o α-α corner o α β barrel- each parallel β segment is attached to its neighbor by an α-helical segment  Complex types- o All α o All β o α / β - the α and β segments are interspersed or alternate o α+β - the α and β regions are somewhat segregated.

Quaternary Structure

 Association of more than one polypeptides  Each unit of this protein is called as a subunit and the protein is an oligomeric protein  Subunits (monomers) can be identical or different.  The protein is homopolymeric or heteropolymeric.  Disulfide bonds usually stabilize the oligomer.

Structure Prediction

 Individual aa have a preference for specific 2D structure o α-helix (default): A, E, L, M, C o β-sheets (steric clash): V, T, I, F, W, Y o Bends: P, G, N  No definite rules for 3D structure. Determined by overall sequence and tertiary interactions between remote residues; decrease in free energy.  Prediction based on computer calculations and comparison to similar domains of known structure

Post Translational Modifications

 During synthesis proteins can incorporate only each of the 20 aa  Many amino acids can be enzymatically modified after incorporation into proteins  Reversible phosphorylation of S, T, Y serve as regulatory switches  Amino-terminal acetylation prevents degradation  Glycosylation and fatty acylation makes proteins respectively more hydrophilic or hydrophobic  Protein stability is enhanced by hydroxylation of P in collagen and carboxylation of E in prothrombin

Functional Proteins 1. Collagen

 Tripeptide repeat of Tropocollagen  Triple helix structure  Left handed alpha chains  3.3 amino acids per turn  Fundamental unit- tropocollagen  3 alpha chains twist in right handed

Unit 1  Tropocollagen- repeats of tripeptide Gly-X-Y  X-proline  Y-3/4 hydroxyproline (prolyl hydroxylase)  5 hydroxylysine( lysyl hydroxylase)

Crosslinking in Collagen

 Intramolecular cross links between ε-amino gps of 2 lysine by lysyl oxidase (Cu dependant enzyme)  Reactive aldehydes of allysine are formed  Side chains of aldehydes get linked covalently  Intermolecular cross linking of tropocollagen

Synthesis of collagen

 Synthesis and entry of polypeptide into lumen of rough ER  Hydroxylation of prolyl and lysyl residues  Glycosylation  Formation of triple helix procollagen  Packaged into transport vescicle- Exocytosis  Cleavage & formation of tropocollagen  Lateral covalent cross linking of tropocollagen  Aggregation of fibrils

2. Elastin

 Highly hydrophobic connective tissue.  Extensibility and elasticity  72KDa tropoelastin  Rich in nonpolar amino acids  High proline and glycine content  No glycosylation  Has hydroxyproline but no hydroxylysine

3. Desmosine

 Lysyl residues of tropoelastin  Lysyl oxidase -oxidative deamination  3 aldehydes+1 lysine  Desmosine formation

4. Alpha-Keratin

 constitute almost the entire dry weight of hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin  Alpha-keratin is rich in the hydrophobic residues Ala,Val, Leu, Ile, Met, and Phe.  The -keratin helix is a right-handed helix  Two strands of -keratin, oriented in parallel (with their amino termini at the same end), are wrapped about each other to form a supertwisted coiled coil.

Figure 1.7.14 Structure of Desmosine

5. Intermediate filament of eukaryotes

 Alpha keratin- mammals  Bete keratin- birds and reptiles  Alpha keratin- hetero dimer of TypeI (acidic) and Type II (neutral or basic)  Super coil is left handed  Two heterodimers antiparallely join to form a tetramer (protofilament)  2 protofilaments- protofibril  4 protofibrils- microfibril  Many microfibrils – macrofibril  Rich in cysteine  Hemoglobin part has been dealt in Respiratory System of Unit 7