UNIVERSITAS BRAWIJAYA Prof. Dr.sc.agr. Ir. Suyadi, MS. PROTEIN Elements: C, H, O, N, (S, P) Kuliah ke-4 BIOKIMIA FAKULTAS PETERNAKAN UNIVERSITAS BRAWIJAYA Prof. Dr.sc.agr. Ir. Suyadi, MS.

INTRODUCTION: PROTEIN Elements: C, H, O, N, and sometimes S. Function: Enzymes, structural proteins, storage proteins, transport proteins, hormones, proteins for movement, protection, and toxins.

Introduction The subunits of a protein are amino acids or to be precise amino acid residues. An amino acid consists of: a central carbon atom (the alpha Carbon Calpha) and an amino group (NH2), a hydrogen atom (H), a carboxy group (COOH) and a side chain (R) which are bound to the Calpha.

Gugus amino dan karboksil

Gugus asam amino

Figure : Peptide bond linking two amino acids

Jenis Protein: Primer, sekunder, tertier, Quarter

General Structure Proteins are made from several amino acids, bonded together. It is the arrangement of the amino acid that forms the primary structure of proteins. The basic amino acid form has a carboxyl group on one end, a methyl group that only has one hydrogen in the middle, and a amino group on the other end. Attached to the methyl group is a R group.

General Structure Proteins are made from several amino acids, bonded together. It is the arrangement of the amino acid that forms the primary structure of proteins. The basic amino acid form has a carboxyl group on one end, a methyl group that only has one hydrogen in the middle, and a amino group on the other end. Attached to the methyl group is a R group.

Macam asam amino

General Structure There are 20+ amino acids, each differing only in the composition of the R groups. An R group could be a sulfydrl, another methyl, a string a methyls, rings of carbons, and several other organic groups. Proteins can be either acidic or basic, hydrophilic or hydrophobic. The following table shows 20 amino acids that common in proteins.

Proteins play key roles in a living system Three examples of protein functions Catalysis: Almost all chemical reactions in a living cell are catalyzed by protein enzymes. Transport: Some proteins transports various substances, such as oxygen, ions, and so on. Information transfer: For example, hormones. Alcohol dehydrogenase oxidizes alcohols to aldehydes or ketones Haemoglobin carries oxygen Insulin controls the amount of sugar in the blood

Amino acid: Basic unit of protein COO- NH3+ C R H Different side chains, R, determin the properties of 20 amino acids. Amino group Carboxylic acid group An amino acid

White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic 20 Amino acids Glycine (G) Alanine (A) Valine (V) Isoleucine (I) Leucine (L) Proline (P) Methionine (M) Phenylalanine (F) Tryptophan (W) Asparagine (N) Glutamine (Q) Serine (S) Threonine (T) Tyrosine (Y) Cysteine (C) Asparatic acid (D) Glutamic acid (E) Lysine (K) Arginine (R) Histidine (H) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

Proteins are linear polymers of amino acids NH3＋ C COOー ＋ NH3＋ C COOー ＋ H H A carboxylic acid condenses with an amino group with the release of a water H2O H2O R1 R2 R3 NH3＋ C CO NH C CO NH C CO H Peptide bond H Peptide bond H The amino acid sequence is called as primary structure F T D A G S K A N G S

Amino acid sequence is encoded by DNA base sequence in a gene DNA molecule DNA base sequence ・ C G A T ＝

Amino acid sequence is encoded by DNA base sequence in a gene Second letter T C A G First letter TTT Phe TCT Ser TAT Tyr TGT Cys Third letter TTC TCC TAC TGC TTA Leu TCA TAA Stop TGA TTG TCG TAG TGG Trp CTT CCT Pro CAT His CGT Arg CTC CCC CAC CGC CTA CCA CAA Gln CGA CTG CCG CAG CGG ATT Ile ACT Thr AAT Asn AGT ATC ACC AAC AGC ATA ACA AAA Lys AGA ATG Met ACG AAG AGG GTT Val GCT Ala GAT Asp GGT Gly GTC GCC GAC GGC GTA GCA GAA Glu GGA GTG GCG GAG GGG

Gene is protein’s blueprint, genome is life’s blueprint DNA Genome Gene Gene Protein Protein

Gene is protein’s blueprint, genome is life’s blueprint Glycolysis network Genome Gene Protein

In 2003, Human genome sequence was deciphered! Genome is the complete set of genes of a living thing. In 2003, the human genome sequencing was completed. The human genome contains about 3 billion base pairs. The number of genes is estimated to be between 20,000 to 25,000. The difference between the genome of human and that of chimpanzee is only 1.23%! 3 billion base pair => 6 G letters & 1 letter => 1 byte The whole genome can be recorded in just 10 CD-ROMs!

Each Protein has a unique structure Amino acid sequence NLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG Folding!

Basic structural units of proteins: Secondary structure α-helix β-sheet Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.

Three-dimensional structure of proteins Tertiary structure Quaternary structure

Hierarchical nature of protein structure Primary structure (Amino acid sequence) ↓ Secondary structure （α-helix, β-sheet） Tertiary structure （Three-dimensional structure formed by assembly of secondary structures） Quaternary structure （Structure formed by more than one polypeptide chains）

Close relationship between protein structure and its function Example of enzyme reaction Hormone receptor Antibody substrates A enzyme enzyme B Matching the shape to A Digestion of A! enzyme A Binding to A

Protein structure prediction has remained elusive over half a century “Can we predict a protein structure from its amino acid sequence?” Now, impossible!

Protein Classification Proteins can be described as having several layers of structure. At the lowest level, the primary structure of proteins are nothing more that the amino acids which compose the protein, and how those proteins are bonded to each other. The bonds between proteins are called peptide bonds, and they can have either single bonds, double bonds, triple bonds, or more holding the amino acids into a protein molecule. At the next level, the secondary structure of proteins, proteins show a definite geometric pattern. One pattern that the protein can take is a helical structure, similar to a spiral staircase. Hair has such a secondary structure. When examined closely, you can see the turns in the proteins of hair molecules. A second geometric pattern is the pleated sheet, where several polypeptide chains go in several different directions. I think of a sheet of paper, or a length of fabric. When viewed closely, silk fibronin, the silk protein, forms such a shape. Skin, although made of more than just proteins, provides another example of a protein with a sheet structure. The following figure shows the pleated sheet secondary structure of silk.

Contoh struktur protein Skin fibroin

Next, we find a tertiary structure to proteins Next, we find a tertiary structure to proteins. Here, we find the three-dimensional structure of the globular proteins, where disulfide bridges puts kinks and bends in the secondary structure. Again thinking about hair, some people have straight hair, some have wavy hair, and some have curly hair. The links and bends in the secondary structure causes the curls in hair. Curly hair has more kinks and bends that wavy hair, and straight hair has very few, if any bends

Contoh struktur protein Psoriasin

At the last, we see the quaternary structure of proteins At the last, we see the quaternary structure of proteins. This the the form taken by complex proteins formed from two or more smaller, polypeptide chains. The polypeptide chains form pieces of a jigsaw puzzle, that when put together form a single protein. Hemoglobin provides a good example, being made from four polypeptide chains.

Contoh struktur protein Hemoglobin

PENJELASAN STRUKTUR DAN FUNGSI PROTEIN

Protein Function in Cell Enzymes Catalyze biological reactions Structural role Cell wall Cell membrane Cytoplasm

Protein Structure

Protein Structure

Model Molecule: Hemoglobin

Hemoglobin: Background Protein in red blood cells

Hemoglobin: Background Protein in red blood cells Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen Red blood cell

Heme Groups in Hemoglobin

Hemoglobin: Background Protein in red blood cells Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen Picks up oxygen in lungs, releases it in peripheral tissues (e.g. muscles)

Hemoglobin – Quaternary Structure Two alpha subunits and two beta subunits (141 AA per alpha, 146 AA per beta)

Hemoglobin – Tertiary Structure One beta subunit (8 alpha helices)

Hemoglobin – Secondary Structure alpha helix

Structure Stabilizing Interactions Noncovalent Van der Waals forces (transient, weak electrical attraction of one atom for another) Hydrophobic (clustering of nonpolar groups) Hydrogen bonding

Hydrogen Bonding D – H A Involves three atoms: Donor electronegative atom (D) (Nitrogen or Oxygen in proteins) Hydrogen bound to donor (H) Acceptor electronegative atom (A) in close proximity D – H A

D-H Interaction Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge Proximity of the Acceptor A causes further charge separation D – H A δ- δ+

D-H Interaction D – H A δ- δ+ Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge Proximity of the Acceptor A causes further charge separation Result: Closer approach of A to H Higher interaction energy than a simple van der Waals interaction D – H A δ- δ+

And Secondary Structure Hydrogen Bonding And Secondary Structure alpha-helix beta-sheet

Structure Stabilizing Interactions Noncovalent Van der Waals forces (transient, weak electrical attraction of one atom for another) Hydrophobic (clustering of nonpolar groups) Hydrogen bonding Covalent Disulfide bonds

Disulfide Bonds Side chain of cysteine contains highly reactive thiol group Two thiol groups form a disulfide bond

Disulfide Bridge

Disulfide Bonds Side chain of cysteine contains highly reactive thiol group Two thiol groups form a disulfide bond Contribute to the stability of the folded state by linking distant parts of the polypeptide chain 

Disulfide Bridge – Linking Distant Amino Acids

Hemoglobin – Primary Structure NH2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- Gly-Gly-Glu-….. beta subunit amino acid sequence

Protein Structure - Primary Protein: chain of amino acids joined by peptide bonds

Protein Structure - Primary Protein: chain of amino acids joined by peptide bonds Amino Acid Central carbon (Cα) attached to: Hydrogen (H) Amino group (-NH2) Carboxyl group (-COOH) Side chain (R)

General Amino Acid Structure H H2N Cα COOH R

General Amino Acid Structure At pH 7.0 H +H3N Cα COO- R

General Amino Acid Structure

Amino Acids Chiral

Chirality: Glyceraldehyde D-glyderaldehyde L-glyderaldehyde

Amino Acids Chiral 20 naturally occuring; distinguishing side chain

20 Naturally-occurring Amino Acids

Amino Acids Chiral 20 naturally occuring; distinguishing side chain Classification: Non-polar (hydrophobic) Charged polar Uncharged polar

Alanine: Nonpolar

Serine: Uncharged Polar

Aspartic Acid Charged Polar

Glycine Nonpolar (special case)

Peptide Bond Joins amino acids

Peptide Bond Formation

Peptide Chain

Peptide Bond Joins amino acids 40% double bond character Caused by resonance

Peptide bond Joins amino acids 40% double bond character Caused by resonance Results in shorter bond length

Peptide Bond Lengths

Peptide bond Joins amino acids 40% double bond character Caused by resonance Results in shorter bond length Double bond disallows rotation

Protein Conformation Framework Bond rotation determines protein folding, 3D structure

Bond Rotation Determines Protein Folding

Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D

Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D τABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D

Ethane Rotation A A D D B B C C

Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D τABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D Three repeating torsion angles along protein backbone: ω, φ, ψ

Backbone Torsion Angles

Backbone Torsion Angles Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2

Backbone Torsion Angles

Backbone Torsion Angles Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2 Dihedral angle φ : rotation about the bond between N and Cα

Backbone Torsion Angles

Backbone Torsion Angles Dihedral angle ω : rotation about the peptide bond, namely Cα1-{C-N}- Cα2 Dihedral angle φ : rotation about the bond between N and Cα Dihedral angle ψ : rotation about the bond between Cα and the carbonyl carbon

Backbone Torsion Angles

Backbone Torsion Angles ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl π electrons and nitrogen lone pair

Backbone Torsion Angles ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair φ and ψ are flexible, therefore rotation occurs here

Backbone Torsion Angles

Backbone Torsion Angles ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair φ and ψ are flexible, therefore rotation occurs here However, φ and ψ of a given amino acid residue are limited due to steric hindrance

Steric Hindrance Interference to rotation caused by spatial arrangement of atoms within molecule Atoms cannot overlap Atom size defined by van der Waals radii Electron clouds repel each other

Backbone Torsion Angles ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair φ and ψ are flexible, therefore rotation occurs here However, φ and ψ of a given amino acid residue are limited due to steric hindrance Only 10% of the {φ, ψ} combinations are generally observed for proteins First noticed by G.N. Ramachandran

Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality

Homologous Proteins: Enterotoxin and Cholera toxin 80% homology

Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality Low sequence similarity implies little structural similarity

Nonhomologous Proteins: Cytochrome and Barstar Less than 20% homology

Sequence Similarity Sequence similarity implies structural, functional, and evolutionary commonality Low sequence similarity implies little structural similarity Small mutations generally well-tolerated by native structure – with exceptions!

Sequence Similarity Exception Sickle-cell anemia resulting from one residue change in hemoglobin protein Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine

Sickle-cell mutation in hemoglobin sequence

Normal Trait Hemoglobin molecules exist as single, isolated units in RBC, whether oxygen bound or not Cells maintain basic disc shape, whether transporting oxygen or not

Sickle-cell Trait Oxy-hemoglobin is isolated, but de-oxyhemoglobin sticks together in polymers, distorting RBC Some cells take on “sickle” shape

Sickle-cell

RBC Distortion Hydrophobic valine replaces hydrophilic glutamate Causes hemoglobin molecules to repel water and be attracted to one another Leads to the formation of long hemoglobin filaments

Hemoglobin Polymerization Normal Mutant

RBC Distortion Hydrophobic valine replaces hydrophilic glutamate Causes hemoglobin molecules to repel water and be attracted to one another Leads to the formation of long hemoglobin filaments Filaments distort the shape of red blood cells (analogy: icicle in a water balloon) Rigid structure of sickle cells blocks capillaries and prevents red blood cells from delivering oxygen

Capillary Blockage

Sickle-cell Trait Oxy-hemoglobin is isolated, but de-oxyhemoglobin sticks together in polymers, distorting RBC Some cells take on “sickle” shape When hemoglobin again binds oxygen, again becomes isolated Cyclic alteration damages hemoglobin and ultimately RBC itself

Protein: The Machinery of Life NH2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- Gly-Gly-Glu-….. “Life is the mode of existence of proteins, and this mode of existence essentially consists in the constant self-renewal of the chemical constituents of these substances.” Friedrich Engles, 1878

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The subunits of a protein are amino acids or to be precise amino acid residues. An amino acid consists of: a central carbon atom (the alpha Carbon Calpha) and an amino group (NH2), a hydrogen atom (H), a carboxy group (COOH) and a side chain (R) which are bound to the Calpha.