Proteins: Significance, Amino Acid Composition (PDF)

## **2. Proteins: Significance, Amino Acid Composition, Classification Of Amino Acids Based On The Chemical Structure And Polarity Of The Side Chains.** ### **Proteins** * Heterobiopolymers * High molecular weight molecules * Compounds of 20 monomers - amino acids * 19 are optically active (L-amin...

## **2. Proteins: Significance, Amino Acid Composition, Classification Of Amino Acids Based On The Chemical Structure And Polarity Of The Side Chains.** ### **Proteins** * Heterobiopolymers * High molecular weight molecules * Compounds of 20 monomers - amino acids * 19 are optically active (L-amino acids) * 1 is optically inactive (glycine) * 1 is cyclic imino acid (proline) ### **Protein Functions** * **Structural** - establishment and maintenance of structure - histones, collagen * **Transport** - hemoglobin, albumin * **Protection and defense** - immunoglobins * **Control and regulation** - hormones and receptors * **Catalysis** - enzymes * **Movement** - actin, myosin * **Storage** - myoglobin ### **A. Amino acids: functions** **Components of:** * Peptides * Proteins * Phospholipids **Neurotransmitters** * Glutamate * Aspartate * Glycine **Precursors of:** * Keto acids * Biogenic amines * Glucose * Nucleotides * Heme, creatine **Transport molecule for:** * NH₂ groups ### **Classification of Amino Acids Based On The Chemical Structure And Polarity Of The Side Chains:** * **Electrically Charged** **(a)** **Acidic** * Aspartic Acid (Asp) * Glutamic Acid (Glu) **(b)** **Basic** * Lysine (Lys) * Arginine (Arg) * Histidine (His) * **Polar Amino Acids With Uncharged R Groups** * Serine (Ser) * Threonine (Thr) * Cysteine (Cys) * Tyrosine (Tyr) * Asparagine (Asn) * Glutamine (Gin) * **Nonpolar** * Glycine (Gly) * Alanine (Ala) * Valine (Val) * Leucine (Leu) * Isoleucine (Ile) * Methionine (Met) * Tryptophan (Trp) * Phenylalanine (Phe) * Proline (Pro) ### **Hydrophobic Amino Acids** * Alanine (Ala or A) * Valine (Val or V) * Isoleucine (Ile or I) * Leucine (Leu or L) * Methionine (Met or M) * Phenylalanine (Phe or F) * Tyrosine (Tyr or Y) * Tryptophan (Trp or W) ### **Hydrophilic Amino Acids** * **Acidic amino acids** * Aspartate (Asp or D) * Glutamate (Glu or E) * **Basic amino acids** * Lysine (Lys or K) * Arginine (Arg or R) * Histidine (His or H) * **Polar amino acids with uncharged R groups** * Serine (Ser or S) * Threonine (Thr of T) * Asparagine (Asn or N) * Glutamine (Gin or Q) ### **Covalent Bonds And Non-Covalent Interactions In The Protein Molecule** * **Peptide bond** is a type of covalent bond between carboxyl group of one amino acid and the amino group of another amino acid * **Secondary structure** - hydrogen bonds, peptide bonds and disulfide bonds * **Tertiary structure** - hydrophobic bonds, hydrogen bonds, ionic bonds, van der walls forces, disulfide bonds * **Quaternary structure** - same as tertiary ### **Characteristic Features Of The Polypeptide Chains** * A series of amino acids joined by peptide bonds form a polypeptide chain and each amino acid unit in a polypeptide is called a residue * A polypeptide chain has polarity because its ends are different with an a-amino group at one end and an a-carboxyl group at the other * A polypeptide chain consists of a repeating part - called the main chain or backbone and a variable part, comprising the distinctive side chains * Polypeptide back is rich in hydrogen-bonding potential - each residue contains a carbonyl group, which is a good hydrogen-bond acceptor, with exception of proline an NH group, which is a good hydrogen-bond donor * In some proteins, linear polypeptide chain is cross-linked, most common are disulfide bonds ### **Primary Structures** * Sequence of amino acids * Covalent peptide bonds * Genetically predetermined * Determines next orders of organization of the proteins ### **A. Peptide Bonds** * Amino acids are joined covalently by peptide bonds, which are amide linkages between a-carboxyl group of one amino acid and the a-amino group of another * Peptide bonds are broken by prolonged exposure to a strong acid or base at elevated temperatures to hydrolyze the bonds ### **B. Resonance** * Resonance structures of the peptide bond ### **C. Peptide Nomenclature** * Each amino acid residue is numbered starting from the N-terminus * The amino acid residues are named in order from N-terminus to C-terminus ### **Determination Of The Amino Acid Composition Of A Polypeptide** * First step in determining primary structure of a polypeptide is to identify and quantitate its constituent amino acids ### **Steps involved** 1. **Hydrolysis**: A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours 2. **Separation**: This treatment cleaves peptide bonds and releases individual amino acids, which can be separated by cation-exchange chromatography 3. **Quantification**: In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached 4. **Elution**: The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity and other characteristics - Each amino acid is released from the chromatography column by eluting with solutions of increasing ionic strength and pH 5. **Quantitation**: The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin - a reagent that forms a purple compound with most amino acids, ammonia and amines 6. **Spectrophotometric Determination**: Amount of each amino acid is determined spectrophotometrically by measuring amount of light absorbed by ninhydrin derivative ### **Sequencing Of The Peptide From Its N-Terminal End** * **Stepwise process** of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end * **Phenylisothicyanate**, known as Edman reagent, is used to label amino-terminal residue under mildly alkaline conditions * **Resulting phenylthiohydantoin derivative** introduces an instability in the N-terminal peptide bond that can be selectively hydrolyzed without cleaving the other peptide bonds * **Identity of amino acid derivative** can then be determined ### **Cleavage Of The Polypeptide Into Smaller Fragments** * **Many polypeptides** have a primary structure composed of more than 100 amino acids * **These molecules cannot be sequenced directly** from end to end * **These large molecules can be cleaved** at specific sites and resulting fragments sequenced * **By using more than one cleaving agent** on separate sample of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, providing a complete amino acid sequence of the large polypeptide * **Enzymes that hydrolyze peptide bonds - peptidases** ### **Determination Of A Protein's Primary Structure By DNA Sequencing** * **Sequence of nucleotides** in a protein-coding region of the DNA specifies amino acid sequence of a polypeptide * **If nucleotide sequence can be determined**, it’s possible to translate sequence of nucleotides into amino acid sequence of the polypeptide ### **Differences In Primary Structure Of Hemoglobin A And S** * Hemoglobin A is the hemoglobin molecule found in normal RBCs during childhood and adulthood * Hemoglobin S is the abnormal variant of hemoglobin A, which occurs in sickle-red blood cells * In Hb-S, both the a-chains have same amino acid sequence as those of normal Hb-A, but in both β-chains glutamic acid in 6th position is replaced by valine ### **Insulin** * Insulin is composed of 51 amino acids arranged in 2 polypeptide chains, which are linked together by 2 disulfide bridges * Insulin molecule contains an intramolecular disulfide bridge between amino acid residues of the A chain * A-chain contains 21 amino acids and B-chain contains 30 amino acids * A-chain, N- terminal amino acid is glycine and C- terminal is asparagine * B-chain, N- terminal amino acid is phenylalanine and C- terminal is threonine * Both chains are held together by disulfidge bridges, Cys 7 and Cys 20 of A-chain are joined to Cys 7 and Cys 19 of B- chain * A chain also carries an intra-chain between Cys 6 and Cys 11 ### **Diabetes Mellitus** * **Type 1**: body’s immune system destroys the cells that release insulin, eliminating insulin production from the body. Without insulin, cells cannot absorb sugar (glucose), which they need to produce energy * **Type 2**: body isn’t able to use insulin correctly - insulin resistance ### **Primary Structure Of Insulin In Different Species** | Species | 8^th | 9^th | 10^th | 30^th | |---|---|---|---|---| | Horse | Thr | Gly | Ile | Ala | | Goat & Cattle | Ala | Ser | Val | Ala | | Man | Thr | Ser | Ile | Thr | | Rabbit | Thr | Ser | Ile | Ser |