Protein Structure PDF

PROTEINS & PROTEIN STRUCTURE Newman Osafo, B.Pharm. Ph.D. Department of Pharmacology, FPPS, CoHS, KNUST. [email protected] Reading materials ✘ Lehninger's Principles of Biochemistry by Nelson DL, Cox MM. 8th Ed. ISBN:...

PROTEINS & PROTEIN STRUCTURE Newman Osafo, B.Pharm. Ph.D. Department of Pharmacology, FPPS, CoHS, KNUST. [email protected] Reading materials ✘ Lehninger's Principles of Biochemistry by Nelson DL, Cox MM. 8th Ed. ISBN: 9781319228002 ✘ Harper’s Illustrated Biochemistry by Kennelly PJ, et al. 32nd Ed. ISBN: 9781264795673 ✘ Biochemistry by Berg JM, Tymoczko JL, Gatto GJJr and Stryer L. 9th Ed. ISBN: 9781319114671 2  OBJECTIVES ✘To know the various levels of protein structure ✗Primary structure ✗Secondary structure Alpha helix – keratin, silk, collagen Beta sheets Beta bends ✗Tertiary structure Domains Protein folding ✗Quaternary proteins Immunoglobulins Myoglobin Hemoglobin and Hemoglobinopathies ✘Denaturation of proteins ✘Protein misfolding ✘Protein degradation 3 OVERVIEW ✘linear sequence of the linked amino acids ✗contains the information necessary to generate a protein molecule with a unique three-dimensional shape ✘Complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels, namely, ✗primary ✗secondary ✗tertiary, and ✗quaternary 4 5/23/2024  Four Levels of Protein Structure Primary Tertiary (Sequence) (Folding by R-group interactions) Quaternary (Two or more chains associating) Secondary (Coiling by Hydrogen Bonding) 5 5/23/2024 PRIMARY STRUCTURE OF PROTEINS 6  PRIMARY STRUCTURE OF PROTEINS ✘This is the sequence of amino acids in a protein ✘The primary structure of a protein is required for ✗an understanding of a protein's structure ✗its mechanism of action at a molecular level ✗and its relationship to other proteins with similar physiological roles ✘Understanding the primary structure of proteins is important because ✗many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function ✗ If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease 7 Besides giving information on the pathway for formation of active insulin, knowledge of primary structures shows the role of amino acids in the structure of insulin through comparison of the primary structures of the insulins from different animal species 5/23/2024 8 Differences in Primary Structure of Insulins Used in Treatment of Diabetes Mellitus ✘Both porcine and bovine insulins are largely no longer used in the treatment of diabetes in humans ✗Compatibility to humans was due to the small number and the conservative nature of the changes between the amino acid sequences of the insulins ✘These changes do not significantly perturb the three-dimensional structure of the insulins from that of human insulin ✘Human insulin, produced by recombinant DNA technology, now available for clinical use ✗It can be made using genetically engineered bacteria or by modifying pig insulin Animal insulin: hypurin (s), hypurin isophane (i), hypurine lente (l), hypurin PZI (l) Human insulin: regular insulin (s), Neutral Protamine Hagedorn (NPH) (i) Human analogue insulin: lispro (r), aspart (r), glargine (l), detemir (l), degludec (ul) 9 SECONDARY STRUCTURE OF PROTEINS 5/23/2024 10  SECONDARY STRUCTURE OF PROTEINS ✘The polypeptide backbone generally forms regular arrangements of amino acids that are located near to each other in the linear sequence - the secondary structure of the polypeptide ✘Examples of secondary structures frequently encountered in proteins are ✗α-helix ✗β-sheet ✗β-bend 11 5/23/2024 α-Helix ✘The most common polypeptide helices found in nature It is ✗a spiral structure ✗consist of a tightly packed, coiled polypeptide backbone core ✗the side chains of the component amino acids extends outward from the central axis to avoid interfering sterically with each other ✗ In this structure the polypeptide backbone is tightly wound around an imaginary axis drawn longitudinally through the middle of the helix ✘Examples of proteins containing α-helices ✗the keratins in hair, skin and porcupine quills their rigidity is determined by the number of disulfide bonds between the constituent polypeptide chains ✗Myoglobin Contains ~ 80% α-helices, it’s a globular and flexible molecule 12 α-Helix ✘Extensive hydrogen bonding between the peptide-bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone stabilizes an α-helix ✗The hydrogen bonds extend up the spiral from the carbonyl oxygen of one peptide bond to the -NH- group of a peptide linkage four residues ahead in the polypeptide ✗This ensures that all but the first and last peptide bond components are linked to each other through hydrogen bonds ✗Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix The structure is stabilized by a hydrogen bond between the hydrogen atom attached to the electronegative nitrogen atom of a peptide linkage and the electronegative carbonyl oxygen atom of the fourth amino acid on the amino-terminal side of that 13 peptide bond Amino Acid Sequence Affects α-Helix Stability Proline disrupts an α-helix because ✗Its amino group is not geometrically compatible with the right-handed spiral of the α-helix ✗Non-rotational N-Cα bond & no substituent H ✘Glycine occurs less frequently because of its conformational flexibility [coiling]. ✘Glutamate, aspartate, histidine, lysine, or arginine also disrupt the helix because ✗they are charged amino acids and form ionic bonds, or repel each other electrostatically ✘Tryptophan, with bulky side chains, or valine or isoleucine, that branch at the β-carbon (the first carbon in the R-group, next to the α- carbon) can interfere with formation of the α-helix if they are present in large numbers 15 β-Sheet ✘ In the β conformation, the backbone of the polypeptide chain is extended into a zigzag rather than helical structure ✘The zigzag polypeptide chains can be arranged side by side to form a structure resembling a series of pleats ✘Hydrogen bonds are formed between adjacent segments of polypeptide chain ✘When illustrations are made of protein structure, β-strands are often visualized as broad arrows 16 β-Sheet ✘Comparing a β-sheet to an α-helix ✗Unlike the α-helix, β-sheets are composed of two or more peptide chains (β- strands), or segments of polypeptide chains, which are almost fully extended ✗Also in β-sheets the hydrogen bonds are perpendicular to the polypeptide backbone 17 5/23/2024 The regular conformation of the polypeptide backbone in the beta sheet. ✘A β-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains that are arranged either ✗Antiparallel to each other (with the N-terminal and C-terminal ends of the β- strands alternating ✗Parallel with all the N-termini of the β-strands together  β-bend/turns ✘Normally present in globular proteins ✘Nearly 2/3rds of the proteins in turns or loops ✘ These are the connecting elements that link successive runs of α-helix or β-conformation. ✘Β-turns connect the ends of two adjacent segments of an antiparallel β-sheets.  Fibrous Proteins Highly elongated protein molecules whose shapes are dominated by a single type of secondary structure Type Example Characteristics 1. Coiled Coil Keratin durable, insoluble, unreactive 2.  Sheet Silk, fibroin of spider web soft, flexible 3. Triple Helix Collagen strong, high tensile strength Keratin Principal component of hair, nails, wool, horns, hooves, scales, feathers, shells  keratin - in mammals  keratin - in birds and reptiles The -keratin chain is an -helix. Pairs of -helix chains are inter-wound to form a two-chain coiled coil. The chains wind in a left-handed sense Each -keratin chain consists of ~310 residues having a 7- residue repeat: a-b-c-d-e-f-g where residues a and d are nonpolar 22 A coiled-coil Collagen - a triple helix Single collagen molecule contains 3 polypeptide chains Each chain is a left-handed helix (3 residues/turn) 3 helical chains are twisted together in a right-handed manner to form a superhelical structure Distinctive amino acid composition 30% Gly and 15-30% Pro or Hyp (hydroxyproline) 24 5/23/2024 Symptoms of Diseases of Abnormal Collagen Synthesis ✘Collagen is present in virtually all tissues and is the most abundant protein in the body ✘Certain organs depend heavily on normal collagen structure to function physiologically ✘Abnormal collagen synthesis or structure causes: ✗dysfunction of cardiovascular organs (aortic and arterial aneurysms and heart valve malfunction) ✗bone (fragility and easy fracturing) ✗skin (poor healing and unusual distensibility) ✗joints (hypermobility and arthritis) ✗eyes (dislocation of the lens) Collagen Disorders 1. Scurvy (vitamin C deficiency) 2. Osteogenesis imperfecta (brittle bone disease) (OI) 25 3. Ehlers-Danlos syndrome Silk -  sheet consists of antiparallel  sheets 6-residue repeat (-Gly-Ser-Gly-Ala-Gly-Ala-)n The  sheets stack to form a microcrystalline array 26 Major Histocompatibility Complex/HLA Model of binding site in class I MHC (major histocompatibility complex) molecules, which are involved in graft rejection A sheet comprising eight antiparallel b strands (green) forms the bottom of the binding cleft, which is lined by a pair of a helices (blue) A disulfide bond is shown as two connected yellow spheres. The MHC binding cleft is large enough to bind a peptide 8 -10 residues long 27 5/23/2024 TERTIARY STRUCTURE OF GLOBULAR PROTEINS 5/23/2024 28  TERTIARY STRUCTURE ✘Polypeptides continue folding beyond the formation of secondary structure ✘Itis only with the complete, compact folding into tertiary (3°) structure that they attain their "native conformation" and become active proteins (as a result of the creation of active sites). 5/23/2024 29 TERTIARY STRUCTURE OF GLOBULAR PROTEINS ✘The primary structure of a polypeptide chain determines its tertiary structure ✘When globular proteins are in aqueous solutions: ✗Their structure is compact, with a high-density (close packing) of the atoms in the core of the molecule ✗Hydrophobic side chains are buried in the interior ✗Hydrophilic groups are generally found on the surface of the molecule ✗All hydrophilic groups (including components of the peptide bond) located in the interior of the polypeptide are involved in hydrogen bonds or electrostatic interactions ✘The α-helix and β-sheet structures provide maximal hydrogen bonding for peptide bond components within the interior of polypeptides ✗This eliminates the possibility that water molecules may become bound to these hydrophilic groups and, thus, disrupt the integrity of the protein 30 Stable protein folding patterns. Adapted from Lehninger’s Principles of Biochemistry Domains ✘Protein Domains are the fundamental functional and three-dimensional structural units of a polypeptide ✗They are modular units from which larger proteins are built ✘The protein domain - a substructure produced by any part of a polypeptide chain that can fold semi-independently into a compact, stable structure ✗Polypeptide chains that are greater than 200 amino acids in length generally consist of two or more domains ✗The different domains of a protein are often associated with different functions ✘The core of a domain is built from combinations of super-secondary structural elements (motifs) ✘Folding of the peptide chain within a domain usually occurs independently of folding in other domains ✗Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain 33 Pyruvate kinase, a protein with three domains Interactions stabilizing tertiary structure ✘The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence ✘Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure ✘Different types of interactions cooperate in stabilizing the tertiary structures of globular proteins 1. Covalent disulfide bonds 2. Hydrophobic interactions 3. Hydrogen bonds 4. Ionic interactions 5. Salt bridges 35 Protein folding ✘Interactions between the side chains of amino acids determine how a long polypeptide chain folds into the intricate three-dimensional shape of the functional protein ✘Protein folding, which occurs within the cell in sec to min, employs a shortcut through the maze of all folding possibilities ✘As a peptide folds, its amino acid side chains are attracted and repulsed according to their chemical properties ✗positively and negatively charged side chains attract each other ✗Similarly charged side chains repel each other ✗In addition, interactions involving hydrogen bonds, hydrophobic interactions, and disulfide bonds all seek to exert an influence on the folding process 36 5/23/2024 Protein folding ✘Cells contain enzymes that facilitate the folding process ✗These include cis-trans prolyl isomerases, protein disulfide isomerases, and chaperone proteins 1. Cis-trans Prolyl isomerases increase the rate of folding by catalyzing inter-conversion of cis and trans peptide bonds of proline residues within the polypeptide chain 2. Protein disulfide isomerases catalyze the breakage and formation of disulfide cystine linkages so incorrect linkages are not stabilized and the correct arrangement of cystine linkages for the folded conformation is rapidly achieved 3. Chaperone proteins were discovered as heat shock proteins (HSPs), a family of proteins whose synthesis is increased at elevated temperatures 37 CHAPERONE ✘The chaperones do not change the outcome of the folding process but act to prevent protein aggregation prior to the completion of folding and to prevent formation of metastable dead end or nonproductive intermediates during folding ✘They interact with the polypeptide at various stages during the folding process ✗They increase the rate of the folding process by limiting the number of unproductive folding pathways available to a polypeptide ✗Some are important in keeping the protein unfolded until its synthesis is finished ✗Some also as catalysts by increasing the rates of the final stages in the folding process ✗Others protect proteins as they fold so that their vulnerable, exposed regions do not become tangled in unproductive encounters ✗protecting hydrophobic regions from forming aggregates in aqueous environment ✗Protect cells exposed to a near lethal temperature rise 38 QUATERNARY STRUCTURE OF PROTEINS 5/23/2024 39 QUATERNARY STRUCTURE OF PROTEINS ✘monomeric proteins - Proteins consisting of a single polypeptide chain ✗If there are 2 subunits, the protein is called "dimeric", if 3 subunits "trimeric", and, if several subunits, "multimeric" ✘Polypeptide chains may be structurally identical or totally unrelated ✘The arrangement of these polypeptide subunits is called the quaternary structure of the protein ✘Subunits are held together by non-covalent interactions ✘Subunits may either function independently of each other, or may work cooperatively ✗Hemoglobin has four subunits ✗Pyruvate dehydrogenase (mitochondrial protein involved in energy metabolism) has 72 subunits 40 5/23/2024 Immunoglobulins (Igs) ✘Consist of 2 heavy and 2 light chains joined by a disulfide bond ✘The variable regions of an L and H chain come together to form the antigen binding site of the immunoglobulin  The heavy and light chains come together to form Fab domains, which have the antigen-binding sites at the ends  The two heavy chains form the Fc domain  The Fab domains are linked to the Fc 41 domain by flexible linkers Myoglobin ✘153 AA residues ✘MW 16,700 ✘eight -helices ✘contains a heme group –iron atom –porphyrin ring system Functions include:  Major physiological role is to facilitate oxygen transport in muscle  Essentially increases oxygen solubility in aqueous solutions  In aquatic mammals, myoglobin also functions to store oxygen (10-fold more in seals and whales)  Reversible binding of O2 to myoglobin (Mb) 42 Haemoglobin ✘intracellular protein in red blood cells ✘physiological function is to transport oxygen ✘Transports Hydrogen ion and carbon dioxide ✘binds oxygen in lungs and releases oxygen into tissues ✘quaternary structure –tetrameric protein –two -subunits and two  subunits - 22 –each subunit contains a heme group –Fe(II) binds O2 with O2 = scarlet no O2 = dark purple 43 Haemoglobin ✘Important to remember that human haemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the haemoglobins ✗Each of is a tetramer, composed of two α-globin-like polypeptides and two β-globin-like polypeptides ✘HbF are normally synthesized only during fetal development ✘HbA2 are synthesized in the adult, although at low levels compared with HbA ✘HbA1c is a glycosylated HbA ✘Embryonic Hb 44 Glycosylated/Glycated Hemoglobin, HbA1c ✘A glycosylated hemoglobin, designated HbA1c, is formed spontaneously in RBCs by combination of the NH2 terminal amino groups of the hemoglobin b chain and glucose ✘The concentration of HbA1c is dependent on the concentration of glucose in the blood and the duration of hyperglycemia ✗In prolonged hyperglycemia the concentration may rise to 12 % or more of the total hemoglobin ✘Patients with diabetes mellitus have high concentrations of blood glucose and therefore high amounts of HbA1c ✘The changes in the concentration of HbA1c in diabetic patients can be used to follow the effectiveness of treatment for the diabetes 45 5/23/2024 Haemoglobinopathies Over 300 variations of amino acid sequences of normal adult haemoglobin (HbA) have been reported Traditionally defined as a family of disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both Haemoglobin variants may function normally or abnormally depending on the nature and position of the substitution Those that can have severe clinical consequences include Sickle-cell anemia (HbS), hemoglobin C disease (HbC) and the thalassemia (alpha and beta) syndromes HbS and HbC result from production of Hb with an altered amino acid sequence whereas the thalassemias are caused by decreased production of normal Hb 46  Haemoglobin variants Name Mutation Effect Hammersmith Phe CD1(42) Ser Weakens heme binding Bristol Val E11(67)  Asp Weakens heme binding Bibba Leu H19(136)  Pro Disrupts the H helix Savannah Gly B6(24)  Val Disrupts the B-E helix interface Philly Tyr C1(35)  Phe Disrupts hydrogen bonding at the 1-1 interface Boston His E7(58)  Tyr Promotes methemoglobin formation Milwaukee Val E11(67)  Glu Promotes methemoglobin formation Iwate His F8(87)  Tyr Promotes methemoglobin formation Yakima Asp G1(99)  His Disrupts a hydrogen bond that stabilizes the T conformation Kansas Asn G4(102)  Thr Disrupts a hydrogen bond that stabilizes the R conformation Deoxyhemoglobin S forms insoluble Glu A6(6)  Val Sickle-cell filaments that deform erythrocytes. (hemoglobin S) anemia Mutant Val on one  subunit interacts in hydrophobic pocket of another  subunit , forming linear polymers. 47 Haemoglobin variants  Sickle Cell Disease ✘Most common hereditary blood disorder ✘Most common of the conditions is sickle cell anaemia (SCA) affecting mainly the black population. ✘In SCA, the Haemoglobin called HbS contains normal -chains but its -chain contain valine instead of glutamate at residue 6, i.e., a hydrophobic amino acid replaces an acidic one. ✘The hydrophobic valine is able to interact with the 85-Phe and 88- leu of an adjacent deoxy HbS 5/23/2024 49 Consequences of the alteration Modificationof the Hb conformation, stacking of 280 million Hb molecules within each erythrocyte altered by the production of fibrous aggregates Change in shape of erythrocytes from a biconcave disc to a crescent or sickle shape on deoxygenation In homozygotes the erythrocytes interact to form clumps, occlusion of capillaries and consequent reduction in blood flow. Organ damage! SCA is characterized by episodes of pain, chronic haemolytic anaemia and severe infections, usually beginning in early childhood ✘In utero testing: chorionic villus sampling, percutaneous umbilical blood sampling, amniocentesis 5/23/2024 50  Under certain conditions such as low O2 levels, RBCs with HbS distort into sickle cells The sickled cells can block small vessels producing microvascular occlusions which may cause necrosis of the tissue 5/23/2024 51 Detection Gel electrophoresis. Because sickle hemoglobin lacks a glutamate, it is less acidic than HbA. Hemoglobin HbS, therefore, does not migrate as rapidly towards the anode as does HbA It is also possible to diagnose sickle-cell anemia by recombinant DNA techniques SCA – Management A combination of fluids, analgesics, antibiotics and transfusions are used to treat symptoms and complications Hydroxyurea, an antitumor drug, has been shown to be effective in preventing painful crises Hydroxyurea induces the formation of fetal Hb (HbF) - a Hb normally found in the fetus or newborn - which, when present in individuals with SCA, prevents sickling 5/23/2024 52 DENATURATION OF PROTEINS ✘Denaturation occurs when a protein loses its native secondary, tertiary, and/or quaternary structure ✘Denaturing does not cause hydrolysis of peptide bonds The primary structure is not necessarily broken by denaturation ✘Denaturing agents include: ✗heat, organic solvents, mechanical mixing, strong acids or bases, detergents, and ions of heavy metals such as lead and mercury ✘Denaturation may, under ideal conditions, be reversible, in which case the protein refolds into its original native structure when the denaturing agent is removed ✗However, most proteins, once denatured, remain permanently disordered ✘Denatured proteins are often insoluble and, therefore, precipitate from solution 53 5/23/2024 ✘The denatured state is always correlated with the loss of a protein's function ✘Loss of a protein's function is not necessarily synonymous with denaturation, however, because small conformational changes can lead to loss of function ✘A change in conformation of a single side chain in the active site of an enzyme or a change in protonation of a side chain can result in loss of activity, but does not lead to a complete loss of the native protein structure 54 5/23/2024  PROTEIN MISFOLDING ✘Protein folding is a complex, trial and error process that can sometimes result in improperly folded molecules ✘These misfolded proteins are usually tagged and degraded within the cell ✘However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age 5/23/2024 55  PROTEIN MISFOLDING ✘Five common examples of protein-misfolding events that can lead to disease: ✗Improper degradation, ✗Mislocalization, ✗Dominant-negative mutations, ✗Structural alterations that establish novel toxic functions, and ✗Amyloid accumulation. 5/23/2024 56 PROTEIN MISFOLDING ✘Deposits of these misfolded proteins are associated with a number of diseases including amyloidoses ✗Accumulation of these spontaneously aggregating proteins, called amyloids, has been implicated in many degenerating diseases--particularly in the neurodegenerative disorder, Alzheimer disease; transthyretin amyloidosis cardiomyopathy. ✘The prion protein (PrP), has been strongly implicated as the causative agent of transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called "mad cow disease") ✗In TSEs, the infective agent is an altered version of a normal prion protein that acts as a "template" for converting normal protein to the pathogenic conformation 57 5/23/2024 TRANSTHYRETIN AMYLOIDOSIS (ATTR) ✘Occurs as a result of misfolding of transthyretin protein produced by the liver. ✗A protein known to carry vitamin A and thyroxine to different parts of the body ✘This produces amyloid fibrils that can deposit in various tissues causing irreversible damage. ✘Mutation in gene coding for TTR can cause structural changes leading to misfolding (hATTR); normal aging process can render ATTR tetramer prone to misfolding (wATTR) ✘3-4% of African Americans are carriers of the most common variant (i.e. Val42Ile) and have high risk of developing ATTR ✘The wild-type ATTR is common in 25% of patients over 80 years TRANSTHYRETIN AMYLOIDOSIS (ATTR) Transthyretin Amyloidosis cardiomyopathy (ATTR-CM) ✘One of the systemic amyloidosi caused by accumulation of tranthyretin fibril in the myocardium. ✘Occurs in 14% of patients with HF; present in 40-60% of patients with AF at time of diagnoses. ✘wATTR-CM is more common, primarily seen in older pateients and has male predominance. Therapies targetting misfolded transthyretin ✘Patisiran and Inotersen which block synthesis of mutated TTR protein by mRNA silencing. Patisiran is not currently approved for used in ATTR-CM; Inotersen is approved for use in hATTR polyneuropathy (with/without cardiomyopathy). ✘Tafamidis and Diflunisal stabilize TTR tetramer to prevent tissue deposition. Tafamidis has been approved for ATTR-CM with diflunisal still under clinical investigation. ✘A combination of Doxycycline and Tauroursodeoxycholic acid given to remove deposited amyloid fibrils.  Degradation of Proteins ✘The concentration of a protein in a cell is controlled by its rate of synthesis and degradation ✘Understanding the processes that control protein degradation is therefore as equally important as an understanding of the processes that regulate protein synthesis ✘Under many circumstances the denaturation of a protein is the rate controlling step in its degradation. Cellular enzymes and organelles that digest proteins "recognize" denatured protein conformations and eliminate them rapidly 5/23/2024 61 Degradation of Proteins ✘Cellshave both extracellular and intracellular pathways for degrading proteins ✘Major extracellular pathway is the system of digestive proteases, which break down ingested proteins to polypeptides in the intestinal tract ✗Endoproteases such as trypsin and chymotrypsin, cleave the protein backbone adjacent to basic and aromatic residues ✗Exopeptidases, sequentially remove residues from the N-terminus (aminopeptidases) or C-terminus (carboxypeptidases) of proteins ✗Peptidases, split oligopeptides into di- and tripeptides and individual amino acids ✘These small molecules are then transported across the intestinal lining into the bloodstream ✘One major intracellular pathway involves degradation by enzymes within lysosomes, membrane-limited organelles whose interior is acidic 62 Assignment What is the Anfinsen’s dogma? How does the study of protein structure disprove it? Identify exceptions if any and repercussions of this anomaly How can you determine the concentration of protein in a given protein sample in the lab? How do you assess the purity of a protein?