PROTEINS Building blocks, structure and function Aim: You will have a clear picture of protein construction and their general properties Reading materials: Compendium in Biochemistry, page 13-49. Microbiology, A1-A12. Variations in GFP result in a rainbow of fluorescent proteins. C&EN 2008 1
Nobel prize in chemistry 2008: Osamu Shimomura, Martin Chalfie and Roger Y Tsien for the discovery and development of the green fluorescent protein, GFP. What are the proteins doing in the cell? Proteins have many functions in the living cell: Catalysis (enzymes) Transport and storage Mechanical support Immune system (antibodies) Movements Nerve signals Control of growth and differentiation This is possible because: LDH Proteins fold into complex three dimensional structures Proteins have chemically functional groups (e.g. -OH, -SH, -COOH, NH 2 ) Proteins can have stiff or flexible parts Proteins can interact with other molecules 2
Proteins are built by 20 different L-amino acids (1) α-carbon H N H 2 R COOH H α-amino acid There are 4 groups on the chiral α-carbon: H COOH (COO - ) NH 2 (NH 3+ ) R (= side chain) The character of the R-group will give amino acids with different: size form charge hydrophobicity hydrogen bonding capacity chemical reactivity Proteins are built by 20 L-amino acids (2) Aromatic Phenylalanine Phe F " Tyrosine Tyr Y " Tryptophane Trp W Aliphatic Glycine Gly G " Alanine Ala A " Valine Val V " Leucine Leu L " Isoleucine Iso I " Proline Pro P Side chain structure 3
Proteins are built by 20 L-amino acids (3) Acidic Aspartic acid Asp D Side chain structure " Glutaminic acid Glu E Amides Asparagine Asn N " Glutamine Gln Q Basic Lysine Lys K " Histidine His H " Arginine Arg R Sulphurus Cysteine Cys C " Methionine Met M Hydroxylic Serine Ser S " Threonine Thr T Peptides - polypeptides H 3 Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4 Amino acid 5 Aminoterminal Carboxyterminal The order of amino acids = primary structure 4
The peptide bond The peptide bond (CONH) is planar and stiff (double bond character) R 1 -group R 2 -group R 1 -group R 2 -group The peptide bond is almost always in TRANS configuration because of steric hindrance The polypeptide spontaneously folds into regular structures The mixture of STIFF ELEMENTS (the peptide p bond) and ELEMENTS WITH LIMITED MOBILITY in the polypeptide enables the formation of regular structures: ο α-helix ο β-structure ο β-turn o loops These structures are examples of SECONDARY STRUCTURES 5
α-helix All NH and CO in the main chain are engaged in hydrogen bonds running PARALLEL to the helix 3,6 Amino acids per turn PILLAR β-structure (β pleated sheet) Antiparallel Parallel Hydrogen bonds BETWEEN the STRETCHED strands WALL 6
β-turn and loops The β-turn has a hydrogen bond between CO on aa i and NH on aa i+3 Enables the polypeptide to turn its heel" Loops do not have exact regular structures t but bt are stable tbl and well-defined anyway What keeps the protein together in a complete three-dimensional structure? NH 3 OOC S S Connecting forces: the secondary structures α-helix β-structure, β-turn and loops ionic i bonds between side chains hydrogen bonds between side chains SS-bridges (SH-groups from two adjacent cysteines) the inside of the protein is predominately hydrophobic 7
List of terms connected to protein structure Primary structure: chain Secondary structure: Tertiary structure: Quaternary structure: The sequence of the amino acids in the polypeptide The interactions between amino acids close in the sequence (example: α-helix, β-structure, β-turn and loops) Interactions between amino acids distant in the sequence, The structure at large" The arrangement of the subunits in a multimeric protein Subunit: A part (= a complete polypeptide) of a multimeric protein Domain: Some proteins have compact parts with a specific function, e.g. a catalytic domain. Is often coded for by an exon. Random coil: Unfolded polypeptide Native protein: The correctly folded polypeptide structure Denatured protein: The protein has lost its natural folding ( e.g. by extensive heating) In some proteins several polypeptide chains are joined together to multimeric units β α β α β α α β Hemoglobin A tetrameric protein consists of 4 subunits = 4 polypeptide chains 8
The structure is determined by the sequence A certain polypeptide sequence always leads to the same structure Why? Random coil Native protein Is not formed is the most stable one = has the lowest energy The folding process is very rapid Random coil 1 s (10 27 år) Native protein = finished, correctly folded protein Local folding and keeping of successful intermediates. Easiest parts first Random coil Amino acids have different propensity to participate in α-helix, β-structure and β-turn Native protein The final adjustment is relatively slow (1 s) Molten globule: Compact structure with most parts in place. Hydrophobic amino acids in the centre. Completed 9
The living cell has proteins which promote the folding In the TEST TUBE the protein folding is often easy (Diluted conditions) In the LIVING CELL the situation is chaotic - Different proteins can get mixed during folding - Erroneous SS-bonds can be formed Therefore, the living cell has means, e.g.: - Chaperones (proteins protecting unfolded parts of the polypeptide chain) - Disulphide isomerases (break up SS-bonds allowing them to recombine to the one with lowest energy) Completed proteins can get new properties by modification Proteins are built by 20 amino acids in the ribosome Some amino acids can be modified later e.g.: - Ser and Thr are phosphorylated (often as a way to control protein activity) - Acetylation of the amino terminal (stabilised against degradation) - Carbohydrates can be bound to till Asn (a more hydrophilic protein) - Fatty acids can be bound to e.g. Cys (a more hd hydrophobic hbi protein) i) - Pro in collagen can become hydroxylated (the structure is stabilised) Sometimes a part of the protein is removed (the protein/the enzyme is activated) 10
Protein purification Overview Alternative 1 Alternative 2 Bacteria Disintegration (grinding/homogenisation) Beef heart Pure protein e.g. human growth hormone a) coarse conditioning i methods TARGET- MOLECULE Homogenate Centrifugation Raw extract b) high resolution methods How can we separate different proteins? Property Method Size Dialysis, Gel filtration Density Centrifugation Charge Hydrophobicity Specific affinity Solubility Ion exchange chromatography, Electrophoresis Hydrophobic chromatography Affinity chromatography Salt precipitation 11
Cells Homogenate Raw extract Salt precipitation More. Pure protein? You must check the result of the purification process! Take samples Run SDS-PAGE Measure protein content Measure enzymatic activity The process should lead to: Fewer number of protein band Higher specific activity (Units/mg protein) SDS PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis) A very common analytic separation method Native protein Denatured protein covered with negatively charged SDS-molecules Heat with SDS-cocktail Polyakrylamide gel between two glass disks - Staining with Coomassie blue + Rawextract Pure protein Standard 12
Protein characterization Overview Molecular size Gel filtration SDS PAGE Mass spectrometry Amino acid sequence Edman degradation 3-D structure NMR X-ray crystallography Enzyme activity MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser hν AH + 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: MH + + A M + AH +. +20 kv Variable Ground Grid Grid 13
Edman degradation works only for the first 50 amino acids! 14
X-ray crystallography 15
Summary Proteins are biopolymers constructed from 20 α amino acids. The 20 amino acids differ in their side chains: polarity and charge Peptides and proteins are formed by linking amino acids together using amide (peptide) bonds. Protein structure: primary, secondary, tertiary and quaternary Protein sequence analysis: Edman degradation Protein purification: chromatography 16