Amino acids and proteins

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Amino acids and proteins

  1. 1. AMINO ACIDS & PROTEINS Centre for Nano science and Technology Course: Introduction to Nano technology. Code: NST 611 Course instructor: Dr. A. Kasi Viswanath. PRESENTED BY ROOPAVATH UDAY KIRAN M.Tech 1st year
  2. 2. Overview of the Presentation AMINO ACIDS TYPES OF AMINO ACIDS PROPERTIES OF AMINO ACIDS PROTEINS STRUCTURE OF PROTEINS PROPERTIES PROTEIN SYNTHESIS PROTEINS AND AMINO ACIDS IN NANO SCIENCE AND TECHNOLOGY
  3. 3. AMINO ACIDS  Amino acids are biologically important organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.  About 500 amino acids are known and can be classified in many ways.  In the form of proteins, amino acids comprise the second largest component (after water) of human muscles, cells and other tissues. Outside proteins, amino acids perform critical roles in processes such as neurotransmitter transport and biosynthesis.
  4. 4. •Aminegroup actslikea base, tendsto be positive. •Carboxyl groupacts likean acid, tendsto be negative. •“R”group isvariable,from1 atom to 20. •Two aminoacidsjoin togetherto form a dipeptide. •Adjacentcarboxyl and aminogroups bond together.
  5. 5. Amino acids having both the amine and carboxylic acid groups attached to the first (alpha-) carbon atom have particular importance in biochemistry.  They are known as 2-, alpha-, or α-amino acids (generic formula H2NCHRCOOH in most cases where R is an organic substituent known as a "side-chain");often the term "amino acid" is used to refer specifically to these. They include the 22 proteinogenic ("protein- building") amino acids which combine into peptide chains ("polypeptides") to form the building blocks of a vast array of proteins.
  6. 6.  Twenty of the proteinogenic amino acids are encoded directly by triplet codons in the genetic code and are known as "standard" amino acids.  The other two ("non-standard" or "non-canonical") are pyrrolysine (found in methanogenic organisms and other eukaryotes) and selenocysteine (present in many noneukaryotes as well as most eukaryotes). For example, 25 human proteins include selenocysteine (Sec) in their primary structure, and the structurally characterized enzymes (selenoenzymes) employ Sec as the Amino acid 2 catalytic moiety in their active sites.  Pyrrolysine and selenocysteine are encoded via variant codons; for example, selenocysteine is encoded by stop codon and SECIS element. Codon–tRNA combinations not found in nature can also be used to "expand" the genetic code and create novel proteins known as alloproteins incorporating non-proteinogenic amino acids. Many important proteinogenic and non-proteinogenic amino acids also play critical non-protein roles.
  7. 7. Cysteine • The AA Cysteine exists as a dimer: cysteine COOHC NH2 CH2 H HS [O] [H] 2 HCOO C NH2 CH2 H S COOHC NH2 CH2 H S cystine a disulfide linkage
  8. 8. Zwitterions • An acid -COOH and an amine -NH2 group cannot coexist • The H+ migrates to the -NH2 group • COO- and NH3 + are actually present, called a “Zwitter ion” • Zwitter ion = compound where both a positive charge and a negative charge exist on the same molecule • AA are ionic compounds • They are internal salts • In solution their form changes depending on the pH
  9. 9. Formation of a Dipeptide Dehydration synthesis
  10. 10. AminoAcid + Amino Acid -->Dipeptide AminoAcid + Dipeptide --> Tripeptide A.A. + A.A. + …..+ Tripeptide --> Polypeptide CCHN R OH CCHN R OH CCHN R OH CCHN R OH CCHN R OH CCHN R OH peptide bonds peptide bonds side chains amino acid residues  Polypeptides
  11. 11. PROTEINS  Proteins are large biological molecules consisting of one or more chains of amino acids.  Proteins perform a vast array of functions within living organisms, like:  Catalyzing metabolic reactions  Replicating DNA  Responding to stimuli and  Transporting molecules from one location to another.  Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors.
  12. 12. • Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. • Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. • Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle.
  13. 13. Proteins - • Proteins make up all living materials
  14. 14. Characteristics of protein: • Contain carbon, hydrogen, oxygen, nitrogen, and sulfur • Serve as structural components of animals • Serve as control molecules (enzymes) • Serve as transport and messenger molecules • Basic building block is the amino acid
  15. 15. •Functions of proteins: 1. Help fight disease 2. Build new body tissue 3. Enzymes used for digestion and other chemical reactions are proteins (Enzymes speed up the rate of a reaction) 4. Component of all cell membranes
  16. 16. PURIFICATION  Proteins may be purified from other cellular components using a variety of techniques such as: • Ultracentrifugation • Precipitation • Electrophoresis • Chromatography  Methods commonly used to study protein structure and function include: • Immunohistochemistry • Site-directed mutagenesis • Nuclear magnetic resonance • Mass spectrometry.
  17. 17. Proteins are linear polymers of amino acids R1 NH3 + C CO H R2 NH C CO H R3 NH C CO H R2 NH3 + C COOー H + R1 NH3 + C COOー H + H2OH2O Peptide bond Peptide bond The amino acid sequence is called as primary structure A A F NG G S T S D K A carboxylic acid condenses with an amino group with the release of a water molecule
  18. 18. Amino acid sequence is encoded by DNA base sequence in a gene ・ C G C G A A T T C G C G ・ ・ G C G C T T A A G C G C ・ DNA molecule = DNA base sequence
  19. 19. Gene is protein’s blueprint, genome is life’s blueprint Gene GenomeDNA Protein Gene Gene Gene Gene Gene Gene GeneGene GeneGene GeneGene Gene Gene Protein Protein Protein Protein Protein ProteinProtein Protein Protein Protein Protein Protein Protein Protein
  20. 20. Protein Synthesis BIOSYNTHESIS
  21. 21. Making a Protein—Transcription • First Step: Copying of genetic information from DNA to RNA is called Transcription. Why? DNA has the genetic code for the protein that needs to be made, but proteins are made by the ribosomes—ribosomes are outside the nucleus in the cytoplasm. DNA is too large to leave the nucleus (double stranded), but RNA can leave the nucleus (single stranded).
  22. 22. • Part of DNA temporarily unzips and is used as a template to assemble complementary nucleotides into messenger RNA (mRNA).
  23. 23. • mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome. • mRNA then goes through the pores of the nucleus with the DNA code and attaches to the ribosome.
  24. 24. Making a Protein—Translation • Second Step: Decoding of mRNA into a protein is called Translation. • Transfer RNA (tRNA) carries amino acids from the cytoplasm to the ribosome.
  25. 25. These amino acids come from the food we eat. Proteins we eat are broken down into individual amino acids and then simply rearranged into new proteins according to the needs and directions of our DNA. • A series of three adjacent bases in an mRNA molecule codes for a specific amino acid—called a codon. • A triplet of nucleotides in tRNA that is complementary to the codon in mRNA—called an anticodon. • Each tRNA codes for a different amino acid. Amino acid Anticodon
  26. 26. • mRNA carrying the DNA instructions and tRNA carrying amino acids meet in the ribosomes.
  27. 27. • Amino acids are joined together to make a protein. Polypeptide = Protein
  28. 28. Chemical synthesis • Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis. • Chemical ligation to produce peptides in high yield. • Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains. • These methods are useful in laboratory biochemistry and cell biology, though generally not for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids. • Synthesized proteins may not readily assume their native tertiary structure. • Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.
  29. 29. Protein Structure • Primary Structure CCHN R OH CCHN R OH CCHN R OH CCHN R OH CCHN R OH CCHN R OH AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 With any 6 of the 20 common AA residues, the number of possible combinations is 20 x 20 x 20 x 20 x 20 x 20 = 64,000,000 (and this is not nearly large enough to be a protein!)
  30. 30. • Primary Structure • A typical protein could have 60 AA residues. This would have 2060 possible primary sequences. 2060 = 1078 This results in more possibilities for this small protein than there are atoms in the universe! • Sometimes small changes in the 1o structure do not alter the biological function, sometimes they do.
  31. 31. Each Protein has a unique structure Amino acid sequence NLKTEWPELVGKSVEE AKKVILQDKPEAQIIVL PVGTIVTMEYRIDRVR LFVDKLDNIAEVPRVG Folding!
  32. 32. Basic structural units of proteins: Secondary structure α-helix β-sheet Secondary structures, α-helix and β- sheet, have regular hydrogen-bonding patterns.
  33. 33.  Secondary Structure  Repeating patterns within a region  Common patterns  helix  pleated sheet  Originally proposed by o Linus Pauling o Robert Corey   helix  Single protein chain  Shape maintained by intramolecular H bonding between -C=O and H-N-  Helical shape o  helix is clockwise
  34. 34.   pleated sheet  Several protein chains  Shape maintained by intramolecular H bonding and other attractive forces between chains  Chains run anti-parallel and make U turns at ends Random Coils  Few proteins have exclusively  helix or  pleated sheet  Many have non-repeating sections called: Random Coils
  35. 35. Tertiary Structure: The Three dimensional arrangement of every atom in the molecule Includes not just the peptide backbone but the side chains as well These interactions are responsible for the overall folding of the protein This folding defies its function and it’s reactivity The Tertiary structure is formed by the following interactions: – Covalent Bonds – Hydrogen Bonding – Salt Bridges – Hydrophobic Interactions – Metal Ion Coordination
  36. 36. Tertiary Structure
  37. 37. Quaternary Structure: – Highest level of organization – Determines how subunit fit together – Example Hemoglobin (4 sub chains) • 2 chains 141 AA • 2 chains 146 AA - Example - Collagen
  38. 38. 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)
  39. 39. Close relationship between protein structure and its function enzyme A B A Binding to A Digestion of A! enzyme Matching the shape to A Hormone receptor AntibodyExample of enzyme reaction enzyme substrates
  40. 40. Denaturation  Denaturation Any physical or chemical agent that destroys the conformation of a protein is said to “denature” it Examples:  Heat (boil an egg) to gelatin  Addition of 6M Urea (breaks H bonds)  Detergents (surface-active agents)  Reducing agents (break -S-S- bonds)  Acids/Bases/Salts (affect salt bridges)  Heavy metal ions (Hg2+, Pb2+) Some denaturation is reversible  Urea (6M) then add to H2O Some is irreversible  Hard boiling an egg
  41. 41. Summary  Proteins are key players in our living systems.  Proteins are polymers consisting of 20 kinds of amino acids.  Each protein folds into a unique three-dimensional structure defined by its amino acid sequence.  Protein structure has a hierarchical nature.  Protein structure is closely related to its function.  Protein structure prediction is a grand challenge of computational biology.
  42. 42. IMPORTANCE OF PROTEINS IN NANO SCIENCE Protein Bio Devices Nano Drug delivery Drug designing Protein based Bio materials Self assembly, Etc.,
  43. 43. Gastrin Releasing Protein (GRP) Receptor Specific Gold Nano-rods General structure of GNR-BBN conjugates.  GNR-BBN conjugates have very high binding affinity toward GRP receptors in cancer cells and can easily enter into the cancer cell.  The selective delivery of GNRs to tumoral region can be achieved by attaching a target-specific protein (BBN). Bombesin (BBN) Gold nanorod (GNR)
  44. 44. Fluorescently-tagged Proteins  Combination of molecular and cell biological studies analyze in vivo localization of proteins expressed with a fluorescent “tag”  Important that “tag” does not interfere with protein activity  Can examine localization of proteins containing different fluorophores
  45. 45. Self-assembly: • Peptide of 16 AA • Alternating polar/nonpolar • Form stable β-strands and β-sheets • Form nanofibers by hydrophobicity • Matrices with high H2O content
  46. 46. Self-assembly: • Charged head group and nonpolar tail • Form nanotubes and nanovesicles • Form interconnected network • Similar to carbon nanotube behavior
  47. 47. Self-assembly: Non-fibrous ECM components • Adhesion proteins • Growth factors • Topography
  48. 48. Protein Engineering  In all cells proteins have:  Enzyme activities  Structural roles  Allows detailed in vitro studies  Proteins can also be made to do useful operations both in vitro and in cells  Protein engineering involves processes that modify or improve proteins  Protein Engineering manipulates protein production, incorporating modifications to “improve” proteins  Recombinant proteins can provide much information about protein function both in vitro and in vivo  Engineered proteins have huge potentials in biotechnology and medicine
  49. 49. Improving Proteins • Quite difficult to improve on activities of proteins for any particular cell – evolution is very efficient! • Can replace mutated (dysfunctional) proteins • Recent advances have tried to make use of novel or uncommon amino acids - Selenocysteine: in a few proteins in all cells (e.g. formate dehydrogenase in bacteria, glutathione peroxidase in mammals) - Pyrrolysine: found in methanogenic group of Archaea
  50. 50. Uncommon Amino Acids • Expansion of genetic code to uncommon amino acids requires several changes in cells: - Specific aminoacyl-tRNA synthetase - Specific tRNA - New metabolic pathways (??) for synthesis of above molecules • Scientists have used similar approaches to incorporate unnatural amino acids • Added one at a time, but over 30 different amino acids have been introduced • Performed for E. coli, yeast, mammalian cells (a) ketone; (b) azide; (c) photocrosslinker; (d) highly fluorescent; (e) heavy atom for use in crystallography; (f) long-chain cysteine analogue

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