05-02-PROTEINS-STRUCTURE-of-PROTEINS_unlocked.pdf

For Your Eyes Only PROTEINS: STRUCTURE of PROTEINS OVERVIEW A. Peptide Bonds - link together the 20 amino acids commonly found in proteins B. Linear Sequence of the Linked Amino Acids - contains the info...

For Your Eyes Only PROTEINS: STRUCTURE of PROTEINS OVERVIEW A. Peptide Bonds - link together the 20 amino acids commonly found in proteins B. Linear Sequence of the Linked Amino Acids - contains the information necessary to generate a protein molecule with a unique 3-dimensional shape C. Structures of Proteins 1. 4 Levels of Organization in Protein Structure a. Primary Structure - the amino acid sequence b. Secondary Structure - the local three-dimensional structure of a polypeptide without regard to the conformations of its side chains  For Your Eyes Only c. Tertiary Structure - the overall three-dimensional structure of an entire polypeptide d. Quaternary Structure - the three-dimensional arrangement of polypeptides in a protein composed of multiple polypeptides PRIMARY STRUCTURE of PROTEINS - linear sequence of amino acids in the polypeptide chain - dictate the unique characteristics of a protein - determines how a protein folds and interacts with other molecules in the cell to perform its function - synthesized from 20 amino acids arranged in a linear sequence determined by the genetic code 1. Importance of the Knowledge of a Protein’s Amino Acid Sequence (Primary Structure) a. Determining its three-dimensional structure and elucidating its molecular mechanism of action b. Comparing sequences of analogous proteins from different species to gain insights into protein function and evolutionary relationships among the proteins and the organisms that produce them c. Developing diagnostic tests and effective therapies for inherited diseases that are caused by single amino acid changes in a protein A. Peptide Bond - join amino acids covalently - amide linkages between the -carboxyl group of one amino acid and the -amino group of another - not broken by - normal handling - heating - high urea concentrations - nonenzymatic hydrolysis requires prolonged exposure to strong acid or base at elevated temperatures 1. Naming the peptide a. Order of Amino Acids in a Peptide - free amino end (N-terminal) is written to the left - free carboxyl end (C-terminal) is written to the right For Your Eyes Only b. Naming of Polypeptide - each amino acid in a polypeptide  “residue” or “moiety” - amino acid name ends (-an, -ine, -ic, -ate) removed + suffix “-yl” except the C-terminal amino acid 2. Characteristics of the Peptide a. Hybrid - peptide bond is a hybrid of two resonance structures, one of which has partial double bond character, so that the carboxyl and amide groups that form the bond must, therefore, remain planar  peptide backbone consists of a sequence of rigid planes formed by the peptide groups i. Partial Double-Bond Character - shorter than a single bond - rigid and planar b. Rotation Around the Bond i. Torsion Angles - rotation within certain allowed angles can occur around the bond between - the -carbon and the -amino group - the -carbon and the carbonyl group ii. Subject to Steric Constraints - maximize the distance between atoms in the different amino acid side chains - forbid torsion (rotation) angles that place the side chain atoms too close to each other - depend on the specific amino acids present - limit the secondary and tertiary structures that can be formed from the polypeptide chain c. Trans Configuration - because of the steric interference of the R groups in the cis configuration  For Your Eyes Only d. Uncharged but Polar - -C=O and -NH groups of the peptide bond neither accept or give off protons over pH of 2.0 - 12.0 - charged groups in polypeptides - N-terminal -amino group - C-terminal  -carboxyl group - ionized groups in side chains B. Determination of the Amino Acid Composition of a Polypeptide 1. Hydrolysis - by strong acid at 110oC for 24 hours - cleaves peptide bonds  release of individual amino acids  separated by cation-exchange chromatography  applied to a column that contains a resin to which a negatively charged group is tightly attached - amino acids bind to the column with different affinities (depending on charges, hydrophobicity, other characteristics) - each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH - separated amino acids contained in the eluate from the column quantitated by heating them with ninhydrin (reagent that forms a purple compound with most amino acids, ammonia, and amines) - amount of each amino acid is determined spectrophotometrically For Your Eyes Only 2. Cation-Exchange Chromatography - separation of released amino acids - mixture of amino acids applied to a column containing resin to which a negatively charged group is tightly attached ( if attached group is positively charged  anion-exchange chromatography) - amino acids bind to the column with different affinities depending on their - charges - hydrophobicity - other characteristics - elution with solutions of increasing ionic strength and pH  sequential release of each amino acid from the column 3. Quantitative Analysis (Use of Amino Acid Analyzer) - separated amino acids quantitated by heating with ninhydrin (forms a purple compound with - most amino acids - ammonia - amines - amount determined spectrophotometrically  For Your Eyes Only C. Sequencing of the Peptide from its N-terminal End 1. Edman Degradation a. Phenylisothiocyanate (PITC) - Edman’s reagent - reacts exclusively with the N-terminal amino group of a polypeptide chain (label the amino-terminal residue under mild alkaline conditions) b. Release of Amino Acid Derivative by Acid Hydrolysis - treating the resulting polypeptide with trifluoroacetic acid releases the N-terminal residue as a thiazolinone derivative, which is converted to a phenylthiohydantoin (PTH)-amino acid that can be identified by chromatography  the procedure is then repeated for the newly exposed N-terminal residue of the polypeptide D. Mass Spectrometry - powerful tool for polypeptide sequencing 1. Electrospray Ionization (ESI) - allows for direct sequencing of short polypeptides (< 25 amino acid residues) by measuring the mass-to-charge ratio of ionic gas-phase peptides E. Cleavage of the Polypeptide into Smaller Fragments - many polypeptides have a primary structure composed of >100 amino acids  cannot be sequenced directly from end to end - large molecules can be cleaved at specific sites  resulting fragments sequenced - use of more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide  overlapping fragments generated that permit the proper ordering of the sequenced fragments  For Your Eyes Only F. Determination of a Protein’s Primary Structure by DNA Sequencing - sequence of nucleotides in a coding region of the DNA specifies the amino acid sequence of a polypeptide - nucleotide sequence  genetic code  translation into amino acid sequence - limitations - unable to predict positions of disulfide bonds in the folded chains - unable to identify the amino acids that are posttranslationally modified G. Amino Acid Substitutions in the Primary Structure 1. Mutations in the Genetic Code - result in proteins with an abnormal amino acid sequences (altered primary structure)  improper folding  loss or impairment of normal function - mutations resulting in single amino acid substitutions can affect the functioning of a protein or can confer an advantage specific to a tissue or a set of circumstances  For Your Eyes Only VARIATIONS in PRIMARY STRUCTURE A. Primary Structure of a Protein 1. Variations - to some degree between species - may vary slightly among individuals - amino acid sequence of a normal functional protein can vary somewhat among - individuals - tissues of the same individual - stage of development 2. Tolerated Variations - if they are - confined to noncritical regions (called variant regions) - conservative substitutions (replace one amino acid with one of similar structure) - confer an advantage 3. Hypervariable Regions - if many different amino acid residues are tolerated at a position, the region is called hypervariable  For Your Eyes Only 4. Invariant Regions - regions that form binding sites or are critical for forming a functional three-dimensional structure are usually invariant regions that have exactly the same amino acid sequence from individual to individual, tissue to tissue, or species to species B. Polymorphism in Protein Structure 1. Cause - mutations in DNA that are passed to the next generation - substitution of one base for another in the DNA sequence of nucleotides (a point mutation) - deletion or insertions of bases into DNA - from larger changes 2. Variation has a Distinct Phenotypic Consequence that - contributes to individual characteristics - produces an obvious dysfunction (a congenital or genetically inherited disease) - increases susceptibility to certain diseases 3. Defective Protein - may differ from the most common allele by as little as a single amino acid that is a nonconservative substitution (replacement of one amino acid with another of a different polarity or very different size) in an invariant region  mutations   affect the ability of the protein to - carry out its function - catalyze a particular reaction - reach the appropriate site in a cell  degraded 4. Other Proteins - variations appear to have no significance 5. Polymorphisms - variants of an allele that occur with a significant frequency in the population - almost one third of the genetic loci appear to be polymorphic 6. Stable Polymorphism - when a particular variation of an allele, or polymorphism, increases in the general population to a frequency of over 1% - ex: sickle cell allele - persistence is probably attributable to selective pressure for the heterozygous mutant phenotype, which confers some protection against malaria C. Protein Families and Superfamilies 1. Homologous Family of Proteins - composed of proteins related to the same ancestral protein 2. Paralogs - groups of proteins with similar, but not identical, structure and function - evolved from the same gene after the gene was duplicated - considered members of the same protein family 3. Divergent Evolution - if a gene has duplicated, one gene can continue to perform original function, and the second copy can mutate into a protein with another function or another type of regulation 4. Protein Superfamily - very large families of homologous proteins - subdivided by name into families of proteins with the most similarity in structure a. Paralogs of a Protein Family - considered different proteins - have different names because they have different functions - all present in the same individual i. Myoglobin and the Different Chains of Hemoglobin - are paralogs and members of the same globin family that have similar, but not identical, structures and functions For Your Eyes Only ia. Myoglobin - intracellular heme protein - present in most cells - stores and transports O2 to mitochondria - single polypeptide chain containing one heme oxygen-binding site - the gene is assumed to have evolved from gene duplication of the - chain for hemoglobin, which evolved from duplication of the -chain ib. Hemoglobin - composed of four globin chains, each with a heme oxygen-binding site - present in red blood cells - transports O2 from the lungs to tissues Homologous Families of Proteins - have the same ancestral protein - arose from the same gene Homologs - includes both orthologs and paralogs A. Orthologs - genes from different species that have evolved from a common ancestral gene as different species developed (ex. human and pork insulin) - retain the same function in the course of evolution B. Paralogs - genes related by duplication within the genome of a single species (ex. myoglobin and hemoglobin) - evolve new functions that may, or may not, be related to the original one  For Your Eyes Only D. Tissue and Developmental Variations in Protein Structure 1. Isoforms or Isozymes (Isoenzymes) of a Protein - within the same individual - may be synthesized during different stages of fetal and embryonic development - may be present in different tissues, or may reside in different intracellular locations - all have the same function - catalyze the same reactions - have somewhat different properties and amino acid structure 2. Developmental Variation a. Hemoglobin Isoforms - example of variation during development b. Hemoglobin - expressed as the fetal isozyme HbF during the last trimester of pregnancy until after birth, when it is replaced with HbA i. HbF - composed of 2 hemoglobin  and 2 hemoglobin  polypeptide chains - much higher affinity for O2 than the adult forms  advantage at the low O2 tensions to which the fetus is exposed ii. HbA - adult hemoglobin - 2  and 2  chains iii. Embryonic Hemoglobin -  and  chains - believed to arise evolutionarily from mutation of a duplicated  gene to produce , and mutation of a duplicate  gene to produce  - much higher affinity for O2 than the adult forms  advantage at the low O2 tensions to which the fetus is exposed 3. Tissue-Specific Isoforms - proteins that differ somewhat in primary structure and properties from tissue to tissue, but retain essentially the same function, are called tissue-specific isoforms or isozymes a. Creatine Kinase Isoforms - each composed of two subunits with 60 to 72% sequence homology - bind to the muscle sarcomere i. M Form - produced in skeletal muscle  MM creatine kinase ii. B Form - produced in the brain  BB creatine kinase iii. Cardiac Isoforms - heterodimer MB - MM dimer iv. Other Creatine Kinase Isozymes iva. Heart Mitochondrial Creatine Kinase ivb. “Universal” Mitochondrial Isoform - found in other tissues (In general, most proteins present in both the mitochondria and cytosol will be present as different isoforms) v. Clinical Significance of Isoforms - useful in diagnosing sites of tissue injury and cell death For Your Eyes Only b. Adenylyl Cyclase - example of proteins involved in the response to hormones and are present as several tissue-specific isoforms that help different tissues respond differently to the same hormone - enzyme that catalyzes the synthesis of intracellular 3’, 5’-cyclic adenosine monophosphate (cAMP) - in human tissues, at least nine different isoforms of adenylyl cyclase are coded by different genes in different tissues - overall sequence homology of 50% - two intracellular regions involved in the synthesis of cAMP are an invariant consensus sequence with a 93% identity  For Your Eyes Only E. Species Variations in the Primary Structure of Insulin 1. Insulin - one of the hormones that are highly conserved between species - very few amino acid substitutions a. Structure - polypeptide hormone of 51 amino acids that is composed of two polypeptide chains b. Synthesis - synthesized as a single polypeptide chain - cleaved in three places before secretion to form the C peptide and the active insulin molecule containing the A and B chains  For Your Eyes Only c. Disulfide Bonds - one intrachain and two interchain disulfide bonds  folding of the A and B chains into the correct three-dimensional structure d. Invariant Residues i. Cysteine Residues - engaged in disulfide bonds ii. Residues that form the surface of the insulin molecule that binds to the insulin receptor GENERAL CHARACTERISTICS of THREE-DIMENSIONAL STRUCTURE - the overall conformation of a protein, the particular position of the amino acid side chains in three- dimensional space, gives a protein its function. A. Descriptions of Protein Structure 1. Major Structural Classification of Proteins a. Globular Proteins - usually soluble in aqueous medium - resemble irregular balls b. Fibrous Proteins - geometrically linear - arranged around a single axis - have a repeating unit structure c. Transmembrane Proteins - consists of proteins that have one or more regions aligned to cross the lipid membrane d. DNA-Binding Proteins B. Requirements of the Three-Dimensional Structure - to enable the protein to function in the cell or extracellular medium of the body - almost every region in the sequence of amino acids, the primary structure, participates in fulfilling one or more of these requirements through the chemical properties of the peptide bonds and the individual amino acid side chains 1. Binding Specificity - that is specific for just one molecule, or a group of molecules with similar structural properties - usually define its role  For Your Eyes Only 2. Rigidity Appropriate to its Function - essential for the creation of binding sites and for a stable structure (i.e., a protein that just flopped all over the place could not accomplish anything) 3. Flexibility and Mobility Appropriate to its Function - enables the protein to - fold as it is synthesized - adapt as it binds other proteins and small molecules 4. Solubility or Lipophilicity - external surface appropriate for its environment (e.g., cytoplasmic proteins need to keep polar amino acids on the surface to remain soluble in an aqueous environment 5. Stability - with little tendency to undergo refolding into a form that cannot fulfill its function or that precipitates in the cell 6. Degradability - when it is damaged or no longer needed in the cell SECONDARY STRUCTURE of PROTEINS - regions within polypeptide chains form recurring, localized structures known as secondary structures 1. Two Regular Secondary Structures - contain repeating elements formed by hydrogen bonding between atoms of the peptide bonds a. -Helix b. -sheet 2. Non-regular Non-repetitive Secondary Structures - do not have a repeating element a. Loops b. Coils c. Bends d. Turns - the rigidity of the peptide backbone determines the types of secondary structure that can occur A. -Helix 1. Characteristics a. Most Common Secondary Structural Element of i. Globular Proteins - ex: hemoglobin - 80% -helical - flexible ii. Fibrous Proteins - ex: keratins - almost entirely -helical - major component of tissues such as hairs and skin - rigidity is determined by the number of disulfide bonds between the constituent polypeptide chains iii. Membrane-Spanning Domains iv. DNA-Binding Proteins For Your Eyes Only b. Structure i. Spiral ia. Right-Handed α-Helix (αR) - one of the most common secondary structures - peptide chain is wound like a screw  each turn of the screw covers approximately 3.6 amino acid residues ib. Left-Handed α-Helix (αL) - mirror image of the αR helix - rarely found in nature ii. Stable Rigid Conformation - that maximizes hydrogen bonding while staying within the allowed rotation angles of the polypeptide backbone iii. Core of the Helix - tightly packed and coiled  maximizing association energies between atoms iv. Trans Side Chains of the Amino Acids - project backward and outward from the helix, thereby avoiding steric hindrance with the polypeptide backbone and with each other For Your Eyes Only c. Formation - formed by strong hydrogen bonds between each carbonyl oxygen atom and the amide hydrogen (N-H) of an amino acid residue located four residues further down the chain 2. Hydrogen Bonds - stabilize -helix - formed between peptide-bond carbonyl oxygen and amide hydrogen - extend up the spiral from the carboxyl oxygen of one peptide bond to the -NH- group of a peptide linkage 4 residues ahead in the polypeptide 3. Amino Acids Per Turn - 3.6 amino acids  For Your Eyes Only 4. Amino Acids that Disrupt an -Helix a. Proline - its imino group is nor geometrically compatible with the right-handed spiral of the - helix - inserts a kink in the chain  disrupt smooth helical structure b. Charged Amino Acids - disrupt helix by forming ionic bonds or electrostatically repelling each other i. Glutamate ii. Histidine iii. Arginine iv. Aspartate v. Lysine c. Tryptophan - bulky side chain d. Amino Acids that Branch at the  Carbon i. Valine ii. Isoleucine B. -Sheet - maximizes hydrogen bonding between the peptide backbones while maintaining the allowed torsion angles  For Your Eyes Only 1. Comparison of -Sheet and -Helix a. -Sheets i. Formation - composed of 2 or more peptide chains (-strands) or segments of polypeptide chains that are almost fully extended - can also be formed by a single polypeptide chain folding back on itself ii. Hydrogen Bonds - perpendicular to the polypeptide backbone - the carbonyl oxygen of one peptide bond is hydrogen-bonded to the nitrogen of a peptide bond on an adjacent strand - optimal hydrogen bonding occurs when the sheet is bent (pleated) to form -pleated sheets     - all of the peptide bond components are involved in hydrogen bonding - when hydrogen bonds are formed between polypeptide backbones of separate polypeptide chains  interchain bonds b. -Helix - hydrogen bonds are within the same strand 2. Parallel and Antiparallel Sheets a. Parallel - if the polypeptide strands run in the same direction (as defined by their amino and carboxy terminals) b. Anti-Parallel - if they run in opposite directions - often the same polypeptide chain folded back on itself, with simple hairpin turns or long runs of polypeptide chain connecting the strands c. Amino Acid Side Chains - alternate between extending above and below the plane of the -sheet i. Parallel Sheets - tend to have hydrophobic residues on both sides of the sheets  For Your Eyes Only ii. Anti-Parallel Sheets - usually have a hydrophobic side and a hydrophilic side 3. Amyloid Protein - fibrous proteins a. Alzheimer’s Disease - amyloid proteins deposited in the brain is composed of twisted -pleated sheet fibrils C. Nonrepetitive Secondary Structures - characterized by an abrupt change of direction - the surface of large globular proteins usually has at least one omega loop, a structure with a neck like the capital Greek letter omega -Bends (Reverse Turns) - reverse the direction of polypeptide chains  compact, globular shape - usually found on the surface of protein molecules - often include charged residues - generally composed of 4 amino acids (proline and glycine are commonly present) - often connect strands of antiparallel -sheets - stabilized by ionic and hydrogen bonds  For Your Eyes Only D. Supersecondary Structures (Motifs) - globular proteins are constructed by combining secondary structural elements (-helix, -sheets, nonrepetitive sequences) - form primarily the core region (interior of the molecule) - connected by loop regions (-bends) at the surface of the protein - produced by packing side chains from adjacent secondary structural elements close to each other - ex: certain of the -strands are connected with -helices to form the  structural motif  For Your Eyes Only TERTIARY STRUCTURE of GLOBULAR PROTEINS A. Interactions Stabilizing Tertiary Structure - 3-dimensional structure of each polypeptide is determined by its amino acid sequence - interactions of the amino acid side chains guide the folding of polypeptide chain  compact structure 1. Disulfide Bonds - covalent linkage - formed from sulhydryl group (-SH) of each of 2 cysteine residues  cystine residue - contributes to the stability of the 3-dimensional shape of the protein molecule 2. Hydrophobic Interactions - amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule  associate with hydrophobic amino acids - amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with polar solvent For Your Eyes Only 3. Hydrogen Bonds - amino acid side chains containing oxygen or nitrogen-bound hydrogen (alcohol groups of serine, threonine) can form hydrogen bonds with electron-rich atoms (oxygen of carboxyl groups or carbonyl groups of peptide bonds - formation of hydrogen bond between polar groups on the surface of a protein and the aqueous solvent enhances the solubility of proteins 4. Ionic Interactions - negatively charged groups [carboxyl group (-COO-) in the side chain (aspartate, glutamate)] can interact with positively charged groups [amino group (-NH3+) in the side chain of lysine]  For Your Eyes Only B. Primary Structure of a Polypeptide Chain - determines its tertiary (folding and final arrangement of the secondary structural elements into an overall three-dimensional conformation) structure C. Three-Dimensional Structure - designed to serve all aspects of the protein’s function - creates specific and flexible binding sites for ligands (the compound that binds) - maintains residues on the surface appropriate for the protein’s cellular location, polar residues for cytosolic proteins, and hydrophobic residues for transmembrane proteins (illustrated with the 2 -adrenergic receptor) 1. Globular Proteins in Aqueous Solutions - compact - hydrophobic side chains are buried - hydrophilic structures on the surface - hydrophilic groups in the interior of the polypeptide are involved in hydrogen bonding or electrostatic interactions a. -Helix and -Sheets - provide maximal hydrogen bonding for peptide bond components within the interior of the polypeptides  water molecules may not bind to hydrophilic groups  prevent disruption D. Domains - the tertiary structure of large complex proteins is often described in terms of physically independent regions called structural domains - fundamental functional and 3-dimensional structural unit of a polypeptide - formed from a continuous sequence of amino acids in the polypeptide chain that are folded into a three- dimensional structure independently of the rest of the protein - chains > 200 amino acids in length consists of 2 or more domains - each domain - small, compact - structurally independent of the other domains in the polypeptide chain - two domains are connected through a simpler structure like a loop  For Your Eyes Only E. Role of Chaperones in Protein Folding 1. Chaperones - polypeptide chain-binding (PCB) proteins (heat-shock proteins) - interact with the polypeptide at various stages during the folding process a. Functions - some keep proteins from unfolded until synthesis is finished - some act as catalyst by increasing the rates of the final stages in the folding process - some protect proteins as they fold b. Molecular Chaperones i. Hsp70 Family of Proteins - family of 70 kD proteins - prevent premature folding of polypeptides as they are being synthesized in the ribosome ii. Chaperonins - form barrel-shaped structures - consist of two types of proteins iia. Hsp60 (GroEL in E. coli) iib. Hsp10 (GroES in E. coli) - cycle of conformational changes in GroEL/ES requiring the hydrolysis of 7 ATP molecules coordinates protein folding in the interior of the GroEL/ES complex - a polypeptide engages in an average of 24 cycles of binding to the GroEL/ES complex before acquiring its native structure iii. Hsp90 Family of Proteins F. Folds in Globular Proteins - relatively large patterns of three-dimensional structure that have been recognized in many proteins, including proteins from different branches of the phylogenetic tree - a characteristic activity is associated with each fold 1. Actin Fold a. Function - ATP binding and hydrolysis - ATP is bound into the middle of the cleft of the actin fold by amino acid residues contributed by domains on both sides  conformational change that closes the cleft  ATP is cleaved to ADP and phosphate  serve the function of the protein b. Proteins with Actin Fold - have very little sequence identity - amount of sequence identity is consistent with their membership in the same fold family - all homologs of the same ancestral protein Classifying proteins into fold families may be useful for drug design. The structure of bacterial protein targeted by a drug can be compared with that of the human proteins in the same fold family so that an antibacterial drug can be designed that more specifically targets the bacterial protein - three-dimensional drawings of their actin folds are almost superimposable i. Actin - (G actin) polymerizes to (F actin which) form the cytoskeleton  For Your Eyes Only ATP binding to G actin  addition of G actin to growing ends of F actin polymers Hydrolysis of bound ATP by G actin subunits  dissociation ii. Hexokinase - catalyzes phosphorylation of glucose 2. Nucleotide-Binding Fold - a fold also can be formed by one domain a. Lactate Dehydrogenase (LDH) - domain 1 alone forms the nucleotide binding fold b. Other Proteins that Bind NAD+ or NADP+ - contain a very different fold from a separate fold family - arise from different ancestral lines and have different structures, but have similar properties and function - believed to be the product of convergent evolution G. The Solubility of Globular Proteins in an Aqueous Environment 1. Globular Proteins - most are soluble in the cell 2. Core of a Globular Domain - built from combinations of supersecondary structural elements - has a high content of amino acids with nonpolar side chains (valine, leucine, isoleucine, methionine, and phenylalanine), out of contact with the aqueous medium  hydrophobic - densely packed to maximize attractive van der Waals forces, which exert themselves over short distances  For Your Eyes Only 3. Charged Polar Amino Acid Side Chains - arginine - histidine - lysine - asparagine - glutamine - generally located on the surface of the protein, where they form ion pairs (salt bridges) or are in contact with aqueous solvent - charged side chains often bind inorganic ions (e.g., K+, PO43- or Cl-) to decrease repulsion between like charges - when charged amino acids are located on the interior, they are generally involved in forming specific binding sites 4. Polar Uncharged Amino Acid Side Chains - serine - threonine - asparagine - glutamine - tyrosine - tryptophan - also usually found on the surface of the protein - may occur in the interior, hydrogen bonded to other side chains 5. Cystine Disulfide Bonds - bond formed by two cysteine sulfhydryl groups - sometimes involved in the formation of tertiary structure - generally not needed H. Tertiary Structure of Transmembrane Proteins 1. Transmembrane Proteins - usually have a number of posttranslational modifications that provide additional chemical groups to fulfill requirements of the three-dimensional structure a. 2 -Adrenergic Receptor i. Domains ia. Membrane-Spanning Domains - -helices with hydrophobic residues exposed to the lipid bilayer ib. Intracellular and Extracellular Domains - on either side of the membrane ii. Helices - clump together so that the extracellular loops form a surface that acts as a binding site (binding domain which is a functional domain) for the hormone adrenaline (epinephrine) - once adrenaline binds to the receptor, a conformational change in the arrangement of rigid helical structures is transmitted to the intracellular domains that form a binding site for another signaling protein, a heterotrimeric G protein (a guanosine triphosphate [GTP]- binding protein composed of three different subunits) iii. Amino Terminus - residues 1 - 34 - extends out of the membrane - has branched high mannose oligosaccharides linked through N-glycosidic bonds to the amide of asparagine - anchored in the lipid plasma membrane by a palmitoyl group that forms a thioester with the SH residue of a cysteine iv. COOH Terminus - extends into the cytoplasm - has a number of serine and threonine phosphorylation sites (shown as blue circles) that regulate receptor activity For Your Eyes Only b. Many Ion Channel Proteins, Transport Proteins, Neurotransmitter Receptors, Hormone Receptors - contain similar membrane-spanning segments that are -helices with hydrophobic residues exposed to the lipid bilayer - rigid helices are connected by loops containing hydrophilic amino acid side chains that extend into the aqueous medium on both sides of the membrane  For Your Eyes Only QUARTERNARY STRUCTURE of PROTEINS A. Definition - is the arrangement and association of polypeptide subunits in a geometrically and stoichiometrically specific manner - many proteins function in the cell as dimers, tetramers, or oligomers, proteins in which two, four, or more subunits, respectively, have combined to make one functional protein B. Noncovalent Interactions - hydrophobic interactions - hydrogen bonds - ionic bonds C. Polypeptide Subunits - many proteins consist of 2 or more structurally identical or totally unrelated polypeptide chains - the subunits combine in the same number and in the same way, because the binding between the subunits is dictated by the tertiary structure, which is dictated by the primary structure, which is determined by the genetic code - subunits may function independently or may work cooperatively - ex: hemoglobin - binding of oxygen to one subunit of the tetramer  increased affinity of the other subunits for oxygen - assembly of globular polypeptide subunits into a multi-subunit complex can provide - opportunity for cooperative binding of ligands (e.g. O2 binding to hemoglobin) - form binding sites for complex molecules (e.g., antigen binding to immunoglobulin) - increase stability of the protein - the prefixes “homo” or “hetero” are used to describe identical or different subunits, respectively, of 2, 3, or 4 subunit proteins (e.g., heterotrimeric G proteins have three different subunits) 1. Protomer - unit structure composed of nonidentical subunits 2. Oligomer - ex: F-Actin - multisubunit protein composed of identical G-actin subunits 3. “Multimer” - is sometimes used as a more generic term to designate a complex with many subunits of more than one type D. Contact Regions 1. Between the Subunits of Globular Proteins a. Contents - closely packed nonpolar side chains - hydrogen bonds involving the polypeptide backbones and their side chains - occasional ionic bonds or salt bridges - subunits of globular proteins are very rarely held together by interchain disulfide bonds, and never by other covalent bonds 2. Fibrous and Other Structural Proteins - may be extensively linked to other proteins through covalent bonds a. Collagen - fibrous protein - polypeptide chains - aligned along an axis - have repeating elements - extensively linked to each other through hydrogen bonds  For Your Eyes Only E. Assembly into a Multisubunit Structure 1. Advantages - increases the stability of a protein - increase in size increases the number of possible interactions between amino acid residues and therefore makes it more difficult for a protein to unfold and refold (many soluble proteins are composed of two or four identical or nearly identical subunits with an average size of approximately 200 amino acids) - the different subunits can have different activities and cooperate in a common function - enable the protein to exhibit cooperativity between subunits in binding ligands or to form binding sites with a high affinity for large molecules - a defective region of a protein (e.g., caused by mistranslation or improper folding) can be easily repaired by replacing the defective subunit - the only genetic information necessary to specify a large protein is that specifying its few different self-assembling subunits - provides a structural basis for the regulation of enzymatic activity 2. Chaperonins - act as templates to overcome the kinetic barrier to reaching a stable conformation QUANTITATION of LIGAND BINDING A. Protein Folding - create a three-dimensional binding site for a ligand 1. NAD+ - for the lactate dehydrogenase domain 1 2. ATP - for Hexokinase 3. Adrenaline - for the 2 adrenergic receptor B. Binding Affinity of a Protein for a Ligand - quantitatively described by its association constant (Ka) 1. Ka - which is the equilibrium constant for the binding reaction of a ligand (L) with a protein (P) - equal to the rate constant (k1) for association of the ligand with its binding site divided by the rate constant (k2) for dissociation of the ligand-protein complex (LP) 2. Kd - dissociation constant for ligand-protein binding - of Ka - the tighter the binding of the ligand to the protein, the higher is the Ka and the lower is the Kd  For Your Eyes Only PROTEIN FOLDING 1. Peptide Bonds - rigid 2. Flexibility Around the Other Bonds - allow an enormous number of possible conformations for each protein - every molecule of the same protein folds into the same stable three-dimensional structure (native conformation) A. Primary Structure Determines Folding - the sequence of amino acid side chains dictates the fold pattern of the three-dimensional structure and the assembly of subunits into quaternary structure - in the cell, not all proteins fold into their native conformation on their own - as the protein folds and refolds while it is searching for its native low energy state, it passes through many high-energy conformations that slow the process (kinetic barriers) - kinetic barriers can be overcome by heat shock proteins, which use energy provided by ATP hydrolysis to assist in the folding process 1. Heat Shock Proteins - named for the fact that their synthesis in bacteria increased when the temperature was suddenly raised - present in human cells as different families of proteins with different activities - ex: hsp70 Proteins - bind to nascent polypeptide chains as their synthesis is being completed to keep the uncompleted chains from folding prematurely - also unfold proteins prior to their insertion through the membrane of mitochondria and other organelles B. Cis-Trans Isomerase and Disulfide Isomerase - also participate in folding 1. Cis-Trans Isomerase - converts a trans peptide bond preceding a proline into the cis conformation, which is well suited for making hairpin turns  For Your Eyes Only 2. Disulfide Isomerase - breaks and reforms disulfide bonds between the -SH groups of two cysteine residues in transient structures formed during the folding process PROTEIN DENATURATION A. Denaturation Through Nonenzymatic Modification of Proteins - lead to a loss of function and denaturation of the protein, sometimes to a form that cannot be degraded in the cell 1. Nonenzymatic Glycosylation - glucose that is present in blood, or in interstitial or intra- cellular fluid, binds to an exposed amino group on a protein  forms an irreversibly glycosylated protein - reaction is nonenzymatic  rate of glycosylation is proportionate to the concentration of glucose present, and individuals with hyperglycemia have much higher levels of glycosylated proteins than individuals with normal blood glucose levels A physician used glycosylated hemoglobin levels, specifically the HbA1c fraction, to determine sustained hyperglycemia over a long period. The rate of irreversible nonenzymatic glycosylation of hemoglobin and other proteins is directly proportional to the glucose concentration. The danger of sustained hyperglycemia is that, over time, many proteins become glycosylated and subsequently oxidized, affecting their solubility and ability to function. The glycosylation of collagen in the heart, for example, is believed to result in a cardiomyopathy in patients with chronic uncontrolled diabetes mellitus. In contrast, glycosylation of hemoglobin has little effect on its function.  For Your Eyes Only 2. Nonenzymatic Oxidation - further modify collagen and other glycosylated proteins in tissues are and form additional cross- links  formation of large protein aggregates referred to as AGEs (advanced glycosylation end-products) B. Protein Denaturation by Temperature, pH, and Solvent - disrupt ionic, hydrogen, and hydrophobic bonds 1. pH - at a low pH, ionic bonds and hydrogen bonds formed by carboxylate groups would be disrupted - at a very alkaline pH, hydrogen and ionic bonds formed by the basic amino acids would be disrupted Proteins are denatured in the gastric juice of the stomach, which has a pH of 1 to 2. Although this pH cannot break peptide bonds, disruption of the native conformation makes the protein a better substrate for digestive enzymes 2. Temperature - increases vibrational and rotational energies in the bonds, thereby affecting the energy balance that goes into making a stable three-dimensional conformation Thermal denaturation is often illustrated by the process of cooking an egg. In the presence of heat, the protein albumin converts from its native translucent state to a denatured white precipitate 3. Hydrophobic Molecules - denature proteins by disturbing hydrophobic interactions in the protein - ex: Long-Chain Fatty Acids - inhibit many enzyme-catalyzed reactions by binding nonspecifically to hydrophobic pockets in proteins and disrupting hydrophobic interactions C. Result - results in the unfolding, refolding, loss of native three-dimensional conformation, and disorganization of the protein’s structure (not accompanied by peptide bond hydrolysis)  insoluble  precipitate from solution - may be reversible in rare cases  protein refolds when denaturing agent is removed D. Agents - heat - mechanical mixing - detergents - organic solvents - strong acids or bases - ions of heavy metals (Pb, Hg) Protein precipitates can sometimes be dissolved by amphipathic agents such as urea, guanidine HCl, or SDS (sodium dodecylsulfate) that form extensive hydrogen bonds and hydrophobic interactions with the protein PROTEIN MISFOLDING 1. Causes of Diseases a. Changes in Protein Structure - may affect the protein’s ability to bind other molecules and carry out its function b. Conformational Changes in Proteins - may affect their solubility and degradability For Your Eyes Only 2. Protein Folding - complex, trial and error process - sometimes result in improperly folded molecules 3. Misfolded Proteins - tagged and degraded - control system imperfection  accumulation and deposition intra- or extracellularly  amyloidosis 4. Steps in Protein Folding  For Your Eyes Only A. Amyloidosis 1. Causes of Misfolding - spontaneous occurrence - gene mutation  altered protein - abnormal proteolytic cleavage of apparently normal protein  unique conformational state  formation of long, fibrillar protein assemblies consisting of -pleated sheets 2. Alzheimer Disease - neurodegenerative disease - characterized by the deposition of amyloid a. Amyloids - spontaneously aggregating proteins - accumulation  implicated in many degenerating diseases particularly neurodegenerative diseases (Alzheimer disease)  For Your Eyes Only b. A amyloid - dominant component of amyloid plaque - peptide of 40 - 43 amino acid residues - when aggregated in -pleated sheet configuration  neurotoxic  cognitive impairment - found in - brain parenchyma - around blood vessels - derived by proteolytic cleavages from larger amyloid precursor protein (single transmembrane protein expressed on the cell surface in the brain and other tissues) c. Abnormal tau Protein - key component of the accumulated neurofibrillary tangles in the brain - blocks the action of the normal counterpart Normal tau Protein  helps in the assembly of the microtubular structure 3. Familial Amyloid Polyneuropathy - neurodegenerative disease - characterized by the deposition of amyloid B. Prion Disease 1. Definition a. Prion - stands for proteinaceous infectious agent 2. Etiology - result from misfolding and aggregation of a normal cellular protein 3. Prion Protein (PrP) - cause neurodegenerative diseases by acting as a template to misfold other cellular prion proteins into a form that cannot be degraded - normally found in the brain - encoded by a gene that is a normal component of the human genome - implicated as the causative agent of - transmissible spongiform encephalopathies (TSEs) - Creutzfeldt-Jakob disease in humans - scrapie in sheep - bovine spongiform encephalopathy in cattle (mad cow disease) a. Normal Form - conformation designated PrPc - no -sheet structure and is approximately 40% -helix - spontaneous refolding of PrP proteins into the PrPSc conformation is prevented by a large activation energy barrier that makes this conversion extremely slow  very few molecules of PrPSc are normally formed during a lifetime b. Disease-Causing Form - has the same amino acid composition (as of the normal form) but is folded into a different conformation that aggregates into multimeric protein complexes resistant to proteolytic degradation - conformation designated PrPSc (sc for the prion disease scrapies in sheep) - substantially enriched in -sheet structure  favours the aggregation of PrPSc into multimeric complexes For Your Eyes Only 4. Prion Disease - acquired either through i. Infection (Mad-Cow Disease) ii. Sporadic or Inherited Mutations (e.g., Creutzfeldt-Jakob Disease) a. Infectious Disease - occurs with the ingestion of PrPSc dimers in which the prion protein is already folded into the high  structure  PrPSc proteins are thought to act as a template to lower the activation energy barrier for the conformational change ( helices of the noninfectious replaced by -sheets of the infectious), causing native proteins to refold into the PrPSc conformation much more rapidly (much like the role of chaperonins)  refolding initiates a cascade  For Your Eyes Only NATURE of PROTEINS A. Function 1. Enzymatic Catalysis - most enzymes are proteins - ex: dehydrogenases kinases  For Your Eyes Only 2. Transport and Storage of Small Molecules and Ions a. Storage Proteins - ferritin - myoglobin b. Transport Proteins - hemoglobin - plasma lipoproteins 3. Structural Elements of the Cytoskeleton - strength and structure to cells - forms the fundamental mechanistic components for intracellular and extracellular movement 4. Structure of Skin and Bone a. Collagen - most abundant protein in the body - give high tensile strength b. Proteoglycans 5. Immunity - antibodies 6. Hormonal Regulation - some hormones are proteins (somatotropin, insulin) - cellular receptors 7. Control of Genetic Expression - activators - repressors - other gene expression regulators - DNA-binding proteins 8. Protective Proteins - blood clotting factors - immunoglobulins 9. Contractile/Motile Proteins - actin - tubulin B. Unique Conformation 1. Specificity 2. Disease States - often related to the altered function of the protein (often due to an anomaly in the protein’s structure) a. Hemoglobinopathies - sickle cell anemia b. Marfan’s Syndrome - due to a single amino acid change in an elastic connective tissue protein (fibrillin) c. Cystic Fibrosis - due to a single amino acid deletion in the ATP-binding domain of a transmembrane conductance regulatory protein that is ATP-dependent PROTEIN CLASSIFICATION A. Solubility in Aqueous Salt Solutions - albumin - histones - globulins B. Overall Shape 1. Globular Proteins - many enzymes - compactly folded, coiled polypeptide chains 2. Fibrous Proteins C. Biologic Functions  For Your Eyes Only OTHER SPECIALIZED SYSTEM of CLASSIFICATION of HIGH MEDICAL IMPORTANCE A. Electrophoretic Mobility at pH 8.6 - 1-lipoproteins - 2-lipoproteins - -lipoproteins B. Sedimentation Behavior in an Ultracentrifuge - chylomicrons - VLDL - LDL - HDL - VHDL C. Immunologic Determination of which Apoproteins are Present  For Your Eyes Only SUMMARY