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LECTURE: AMINO ACIDS & PROTEINS Amino acids – structures & properties. Peptides. Proteins –  primary, secondary, tertiary & quaternary structures.  protein folding Proteins have different overall shapes (conformations) Proteins group based on solubility & shape: Fibrous proteins Globular proteins Membrane proteins Proteins in different locations ..\..\hemolysis.mov Rbc ‘ghost’ Hypotonic medium RED BLOOD CELLS contain many types of proteins SOLUBILIZE WITH DETERGENT SOLUBILIZE WITH DETERGENT Cathode (-) Load samples Buffer Gel (Polyacrylamide [PAGE]) DECREASE IN SIZE Anode (+) Buffer Steps in polyacrylamide gel casting to sample loading Casting gel Ready for sample Gel ready for electrophoresis Loading samples Run gel (electrophoresis) Structure of RBC is maintained by many proteins arranged in 3-D. ELECTRON MICROGRAPH: proteins arrangements in the inner membrane of RBC Modified Karp Cell membranes contain proteins of different topologies. INTEGRAL PROTEINS PERIPHERAL PROTEINS: Proteins that are associated with the membrane Receptors, transporters LIPID ANCHORED PROTEINS: Proteins linked via lipids to the membrane Intracellular proteins Extracellular proteins Modified Karp Globular shaped Proteins       Tightly folded into compact globular shape with hydrophobic residues inside and hydrophilic residues on surface. Structurally complex - contains several types of 20 structures. Soluble in aqueous medium. Diffuse readily. Mobile or dynamic functions. Enzymes, transport proteins, antibodies. Fibrous shaped Proteins      Insoluble in aqueous medium Extended structures with hydrophobic residues on the outside Consist largely of one type of 20 structure Structural or protective role a-keratin, collagen, fibroin (silk) REPEATIVE UNITS OF : Gly-X-Pro, or Gly-X-HyPro, Where X is any amino acid Some Proteins are modified  Simple protein contains only polypeptide chain (sometimes with modified amino acids). Some proteins have other modifications - Conjugated proteins (polypeptide chain + prosthetic group – organic or inorganic moiety). Classification based on nature of prosthetic group   Structural Organization of Proteins 4 levels of organisation: Primary, secondary, tertiary & quaternary Secondary structure: local folds Tertiary structure: global folding Quartenary structure: aggregation of global folds Primary structure: linear arrangements Modified Nelson & Cox PRIMARY SEQUENCES  Refers to number and sequence of amino acids number - amino acid composition sequence - amino acid sequence Also location of S-S bond(s), if any.  Note- do not show 3D or local structures Insulin – 2 linear sequences (chains A & B) linked by disulphide bonds. Glia Cell-line Derived Neurotrophic Factor (GDNF) – different way to visualize a protein by bioinformatics Cartoon representation Wire-frame Ball-stick animation Proteins have specific conformations – Important for function Linear PRIMARY sequence of modified GDNF containing C-terminal 134 amino acids. MSPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCEAAETMYDK ILKNLSRSRRLTSDKVGQACCRPVAFDDDLSFLDDSLVYHILRKHSAKRCGCI Secondary structures -Turns -Sheets Loops -Helix Various non-covalent forces dictate and stabilize protein structure Hydrophobic residues buried inside molecule (shielded from the environment) H-bonds Hydrophobic interactions Electrostatic interactions (ionic) Van der Waals interactions Hydrophobic interactions of different residues hold molecule in shape Hydrophilic interactions with water – H bonds The R-group of side chains contributes to these forces. 3-D structure of polypeptides Delocalized bonds result in shorter bond length and result in a rigid bond. Amide plane 2 degree of rotation -  and  angles Tetrahedral bonds of -Carbon Fully extended dipeptide (proteins are not fully extended structures) Steric hindrance limits the number of conformations about -Carbon Most combinations of  &  angles are sterically forbidden. Protein structures are constrained by the amide planes N- terminus C- terminus Bond is not free to rotate Modified Nelson & Cox Calculate the  &  angles for every residue – Ramachandran plot (conformation plot) Ramachandran Plot: shaded portion are favorable angles +180  (deg) 0 -180 -180 0 +180  (deg) Actual angles measured & Ramachandran plot +180 (deg) Cytochrome C 0 (deg) -180 -180 0 +180  (deg)  (deg) Plastocyanin  (deg)  (deg) Because of the constraints of how the amide planes can “twist”, there MUST only be a limited number of favorable secondary structures. Few types of secondary structures are stable and occur widely in proteins -  helices and  sheets Side view Front view Animation. ..\..\..\rasmol\rw32b2a.exe SECONDARY STRUCTURES–most prominent: -helix and -sheet Conformation stabilized largely by weak interactions  Rod-like structure H-bonding  R-groups extend outward in a helical array  Intra-H bond spacing is 4 residues apart  3.6 amino acid residues/turn  Proline residues interrupt a-helix  Commonly found in globular proteins Lodish et al Helix and amino acids Proline generally disrupts a-helix formation because of the backward twist of the gp.destabilizing kink. Hence, proline is seldom found in helices but at the start of helices. Glycine is too flexible and prefer to adopt a random coil structure. -SHEET      Extended “sheet-like” structure Intermolecular H-bonds Parallel or antiparallel -sheets Favour amino acids with compact R groups -(Gly-Ser-Gly-Ala-Gly-Ala)n- sequence motif in the silk protein, fibroin -sheet antiparallel -SHEET CAN BE PARALLEL OR ANTIPARALLEL C O PARALLEL H N ANTIPARALLEL OTHER STRUCTURES Helices/-sheets: ~50% of regular 20 structures of globular proteins Remaining : coil or loop conformation Examples : reverse turns, -bends (connect successive strands of antiparallel -sheets  -Turns   Coil or Loop Usual to find proline and glycine in -turns. Frequency of amino acids in secondary structures TRIPLE HELIX Limited to tropocollagen molecule ( 3 strands of molecules subunit)    Sequence motif of –(Gly-X-Pro/Hypro)n3 left-handed helices wound together to give a right-handed superhelix. Stable superhelix : glycines located on the central axis (small R group) of triple helix. One interchain H-bond for each triplet of amino acid – between NH of Gly and CO of X in adjacent chain. C   N O C C Structural Organization of Proteins 4 levels of organisation: Primary, secondary, tertiary & quaternary Secondary structure: local folds Tertiary structure: global folding Quartenary structure: aggregation of global folds Primary structure: linear arrangements Modified Nelson & Cox SUPER-SECONDARY STRUCTURES (MOTIFS OR FOLDS)  In large proteins, some recognizable structures observed – groups of unique combinations of folds (secondary structures). Combinations of a few secondary structures (helices and -strands) with specific geometric arrangements in some proteins Four classes:  All   All   / ( and  segments are interspersed or alternate)   &  ( and  regions are somewhat segregated)  Number of folds are finite (< 1,000 different types of motifs in all proteins). -helical   -Sheets / motif– interspersed structures of &  +  motif.  and  are segregated and distinct. Examples..cro;1E MG; NS3,epo DOMAINS (modules) – combinations of motifs  Many large proteins contain several discreet, independently folded globular units – domains Each domain typically consists of about 100-200 aa residues and a combination of motifs Have a specific function has two distinct domains, and NAD+ binds to the first domain while G-3-P, the second.   E.g., Glyceraldehyde-3-phosphate dehydrogenase , for example, Glyceraldehyde-3-phosphate dehydrogenase tetramer with a total molecular weight of 145 kD Domain (/ motif) binds glyceraldehyde-3phosphate (not shown) Domain binds NAD+ Many domains are independent structural units and often have distinct functions Some examples of protein domains/modules Complement control domain Immunoglobulin domain Fibronectin type I domain Growth factor domain Kringle domain Domains/modules making up some proteins Immunoglobulin domain Complement domain Epidermal growth factor-like domain -carboxyglutamate residue domain Fibronectin domain Kringle domain QUATERNARY STRUCTURE Stabilize by non-covalent interactions between subunits Multimeric proteins can have 2 – 100’s of subunits For example, mammalian haemoglobin has 2 (141 aas) and 2 (146 aas) polypeptide chains but earthworm Hbs have > 100 subunits If subunits are composed of a number of nonidentical subunits, overall structure is asymmetric and quite complicated If identical subunits or repeating groups of nonidentical subunits, usually symmetrical      Structure-rasmol TETRAMERIC STRUCTURE OF HEMOGLOBIN QUATERNARY STRUCTURE: ADVANTAGES  Oligomers more stable than dissociated subunits – prolong life of protein in vivo. Active sites formed by residues from adjacent chains Error of synthesis is greater for longer/bigger chains.   Therefore make smaller once and the assemble them together.  Subunit interactions : cooperativity/ allosteric effects – function of single subunit is influenced (positively or negatively) by other associating subunits. PROTEIN FAMILIES Proteins sharing similar primary sequences and/or have similar structures/functions – same family. C-type cytochrome of different species – same protein family (homologues) • same functions (electron carrier) • low primary sequence similarity • structurally similar (specially in the interior of the protein) Cytochrome C – sequence & phylogenetic comparisons No. of amino acid differences STRUCTURE-FUNCTION: COLLAGEN (AN EXAMPLE)      Most abundant protein (25-35% of total protein in mammals) Found in bones, tendons, skin, blood vessels, and cornea of the eye (different structures and textures) ~30 genes encode collagen. >19 distinct types of collagen in human. Tropocollagen (basic structural unit of collagen) is a triple helix of 3 polypeptides (1050 aa residues each) Sequence motif of chain: Gly-X-Pro/Hypro Undergoes extensive post-translational modifications Hydroxylation of Pro and Lys residues (requires vitamin C/hydroxylase enzyme) Hydoxyproline – additional H-bond for stability of helix structure Hydroxylysine – attachment sites for carbohydrate moieties (glycosylation)     Collagen – a commonly used biochemical in tissue engineering of bones. Cosmetics. Organization of collagen Collagen fibrils Arrangements of triple helices 3 helices (triple helix) wound together Each helix MODIFICATION OF PROLINE IN COLLAGEN (Vitamin C) OTHER MODIFICATIONS IN COLLAGEN  Oxidation of side chains of (NH2)Lys to form (OH)allysine (an aldehyde) (OH)Allysine-lysine x-links : intermolecular covalent x-links between tropocollagen molecules Allysine-allysine x-links : intramolecular covalent x-links within a tropocollagen unit (unlike keratin in hair where disulphide forms (cys) x-links, resulting in strong fibrils). Essential for formation of strong collagen fibrils Collagen diseases : mutation of glycine is lethal (gly-X-pro) Covalently X- linked     Diseases associated with collagen  Osteogenesis imperfecta results in abnormal bone formation in babies Ehlers-Danlos syndrome is characterized by hyperextensibility of skin, abnormal tissue fragility, and increased joint mobility. Scuvy (not a genetic disease) – affects collagen xlinking. Deficiency of ascorbic acid. Bleeding in gums, poor wound healing. Both can be lethal: due to substitution of a Cys and Ser residue respectively for a Gly (different Gly for each) in collagen. This substitution (especially in C-terminus) produces catastropic effect since it disrupts the Gly-X-Pro (repeat) helical structure of collagen ED syndrome     PROTEIN DENATURATION Loss of biological activity Conformational/structural change Heat, pH, organic solvents, urea, guanidine hydrochloride Destruction of 2o, 3o and 4o structures, but 1o structure remain intact Ease of proteolytic digestion, aggregation Reversible or irreversible DENATURATION OF RIBONUCLEASE A Enzyme (124 aa) – digest RNA. Denatured by: 6 – 8M urea + 2 mercaptoethanol (reducing agent) No activity Two routes for dialysis: either to remove urea or 2mercaptoethanol first Removal of urea first led to recovery of activity       Information for folding resides in primary structure of the protein To unravel protein (re)folding is a major area of research (Ribonuclease A study won Anfinsen a Nobel prize 1972).  DENATURATIONRENATURATION OF RIBONUCLEASE A  8 Cys.  Probability of any 4 Cys forming the native form of disulphide randomly- 1:105.  Renaturation results in fully active protein – refolding results in mostly correct native form of the protein.  Conclusion: primary sequence dictate 3-D structure for this protein. RECOMBINANT INSULIN - BIOTECHNOLOGY  Originally isolated from pig pancreas (1920s)- 1 pancreas/patient/3 days.  slightly immunogenic – human raise antibody to pig insulin.  Human insulin: 1970s advent of molecular biology – cloned human insulin. Cellular processing of insulin B-chain (30aa) A-chain (21aa) C-chain (34aa) Enzymatic cleavages preproinsulin proinsulin Insulin – folded correctly STRATEGY IN PRODUCTION OF HUMAN INSULIN  Produced in bacteria (E.Coli)  Initially produced by genetic engineering of A and B chains separately and then denature/renature to form the correct disulphides.  Co-express and folding of insulin A/B chain in the presence of the C- chain followed by purification.  Folding of a mature polypeptide (e.g., insulin with A-B chain) can be assisted by the presence of polypeptides (e.g., chain of insulin). Genetic engineering to produce proinsulin Denaturation/refolding/oxidize Enzymatic cleavages In vitro. proinsulin Insulin – folded correctly PROTEIN FOLDING PROCESS     Not a random process A 100 residue protein : random search of all  possible conformations will take 1087 seconds  (many times the age of the universe) A cooperative and sequential process :  formation of one part of the structure leads  to the formation of the remaining structure Folding pattern and final conformation  depend on primary structure Observation: process of denaturation, can identify “molten globule” (intermediate state) ENERGETICS IN FOLDING Least stable Most stable Conditions for refolding in vitro Buffer + denaturant + reducing agent (e.g., dithiotheritol) Low temperature Large volume (low concentration of denatured protein; < 0.1 mg/ml) Slow Dialysis to remove denaturant and allow gradual refolding Providing an environment that allows individual molecule to refold. No crowding! PROTEINS ARE FOLDED CORRECTLY INSIDE CELLS Nucleus Nucleus mRNA exporting into the cytoplasm Rough Endoplasmic Rough Endoplasmic reticulum (RER) reticulum (RER) Some proteins are translated and stay in the cytoplasm Inside of the RER PROTEINS FOLDING IS ASSISTED BY CHAPERONES MOLECULAR CHAPERONES –folding machinery inside cells. “ How crowded is the cell?”     Unfolded proteins usually possess numerous solvent-exposed hydrophobic regions Tendency to self-aggregate especially in concentrated solution (e.g, inside a cell) Function to prevent or reverse such improper associations by binding to these hydrophobic areas Subsequent release of these proteins will facilitate proper folding based on their amino acid sequence. These include peptidyl prolyl cistrans-isomerases (PPIs) and protein disulphide isomerases (PDIs) PPIs: catalyze the isomerization of incorrect Xaa-Pro bonds which is a slow rate determining step PDIs: facilitates the formation of correct set of S-S bonds Molecular chaperone    Model of hsp60 family of molecular chaperone Protein folding & Denaturation in vivo (inside a cell) Molecular chaperones (specialized proteins) help fold proteins inside cells. Misfolding can result in death of cells – E.Coli deal by forming inclusion bodies. Mammalian cells cannot form insoluble products inside. They then commit suicide. Quality control in protein synthesis Unfolded protein response (UPR) – transcriptional activation of stress proteins (chaperones) Unfolded & misfolded proteins Native proteins Aggregated proteins Molecular chaperones Molecular chaperones Ubiquitin (76 aa protein) + ATP Ubiquitinated protein Released protein UBIQUITINATION UBIQUITINATION SYSTEM SYSTEM Protein remodelling (refolding) ATP Proteosome Ubiquitin released (Nobel prize – 2004) Degraded protein MISFOLDING CAN RESULT IN DEATH OF CELLS e.g., Prion, Alzheimer’s disease & other neurodegenerative diseases. Prion (proteinaceous infectious agent) Diseases [Transmissible spongiform encephalopathies (TSEs)] Kuru was once found among the Fore tribe in Papua New Guinea. (“laughing deaths” – eating the dead ritual). Misfolded protein ‘cause’ several rare degenerative brain diseases. ‘Mad cow disease’ outbreaks – cow feeds. Fatal. Madcow.mov Single copy gene in chromosome 20 – natural form PrPc.   Prion (protein), PrP, has a molecular mass of 28,000 dalton. Found in the brain tissue of all mammals, but function unknown. 3  helices + 2 short antiparallel  sheets Mutation Altered form – insoluble fibrils Protease sensitive Protease insensitive Misfolding of protein    Cys to Alanine mutation in myostatin  protein results in misfolding of the protein The misfolding leads to inactivation of the  myostatin activity Inactivation of myostatin results in  increased muscle growth  Myostatin protein mutation