Globular and Fibrous Proteins Handouts PDF

Chapter 4: Globular and Fibrous Proteins Nükhet Aykın-Burns, PhD Division of Radiation Health Pharmaceutical Sciences Biomed II, Room 441A-...

Chapter 4: Globular and Fibrous Proteins Nükhet Aykın-Burns, PhD Division of Radiation Health Pharmaceutical Sciences Biomed II, Room 441A-2 LEARNING OBJECTIVES: At the end of this lecture, students should be able to: 1. Explain the main functions of globular and fibrous proteins. 2. Describe different types of folds in globular and fibrous protein structures and give examples 3. Describe the structure and function of myoglobin and hemoglobin 4. Explain the process and factors that effect oxygen binding to heme proteins especially hemoglobin 5. Explain the allosteric effects of hemoglobin 6. Explain CO poisoning 7. Describe minor hemoglobins and their basic functions 8. Describe abnormal hemoglobins in humans as well as major hemoglobinopathies. 9. Describe the structure and function of immunoglobins in general. 10.Describe collagen structure, synthesis and degradation. 11.Explain collagen and elastin related diseases.  Globular Proteins ü Spherical like shaped proteins ü They are mostly water-soluble CORE Val, Leu, Ile, Met, Phe SURFACE Arg, Lys, His, Met, Asp Globular protein : spherical like shaped proteins that are somewhat water- soluble (they form colloids in water), e.g. hemoglobin § In general, the core of a globular domain has a high content of amino acids with nonpolar side chains (e.g. valine, leucine, isoleucine, methionine, phenylalanine), out of contact with aqueous medium (the hydrophobic effect) § This hydrophobic core is densely packed to maximize attractive van der Waals forces (effective in short distances) § The charged polar amino acid side chains (e.g. arginine, histidine, lysine, aspartate, glutamic acid) are generally located on the surface of the protein, where they form ion pairs (salt bridges, ionic interaction) or are in contact with aqueous medium. § Charged side chains often bind inorganic ions (e.g. K+, PO43-, Cl-) to decrease repulsion between like charges. § When charged amino acids are located on the interior part of the protein, they are generally involved in forming specific binding sites. § The polar uncharged amino acid side chains of serine, threonine, asparagine, glutamine, tyrosine, tryptophan are usually found on the surface of the protein, but they sometimes occur in the interior by making hydrogen bonds to other side chains. Functions and Examples of Globular Proteins Storage of ions and molecules – myoglobin, ferritin Transport of ions and molecules – hemoglobin, serotonin transporter Defense against pathogens – antibodies, cytokines Muscle contraction – actin, myosin Biological catalysis – chymotrypsin, lysozyme  Folds in Globular Proteins; Actin Fold For adenosine triphosphate (ATP) binding and hydrolysis First identified in G-actin § Folds in globular proteins are relatively large patterns of 3D structure that can be recognized in many proteins as binding sites. § Actin Fold: Adenosine triphosphate (ATP) binding and hydrolysis § Example: In G-actin, all 4 subdomains contribute a folding pattern called the actin fold, named for the first protein in which it was described. § ATP is bound into the middle of the cleft of the actin fold by amino acid residues contributed by domains on both sides. § ATP binding promotes a conformational change that closes the cleft. Once it is bound, ATP is cleaved to adenosine diphosphate (ADP). § The actin fold is found in proteins as diverse as actin, which polymerizes to form the cytoskeleton, heat-shock protein 70, which uses ATP energy in changing the conformation of other proteins, and hexokinase, which catalyzes phosphorylation of glucose. In all these proteins, ATP binding results in large conformational changes that contribute to the function of the protein.  Folds in Globular Proteins; Nucleotide Binding Fold LDH § A fold can also be formed by one domain. § Example: Lactate dehydrogenase, LDH domain 1 alone forms the nucleotide binding fold. § This fold is a binding site for NAD+ or, in other proteins, molecules with a generally similar structure (e.g. riboflavin). § However, many proteins that bind NAD+ or NAD+ phosphate (NADP+) contain a very different fold from a separate fold family.  Catalase Globular Hemeproteins Cytochrome Heme Hemoglobin § Heme is a complex of protoporphyrin IX and Fe2+. § Heme proteins contain as a tightly bound prosthetic group. § Organic ligands that are tightly bound to proteins, such as heme of myoglobin, are called prosthetic groups. § A protein with its attached prosthetic group is called holoprotein; without the prosthetic , it is called apoprotein. § The 3-dimensional structure of the protein will determine the role of the heme group. § Examples: Cytochromes: heme is an e- carrier & is oxidized & reduced Catalase: it is part of the active site that breaks down H2O2. Hemoglobin & myoglobin: reversibly binds O2.  Myoglobin: Structure and Function Nonpolar crevice ü heart and skeletal muscle ü a reservoir and carrier of oxygen ü α- helixes terminated by proline or β-bends & loops § Myoglobin (Mb) is a compact heme protein found in heart and skeletal muscle. § It functions as a reservoir and carrier of oxygen. § It is a single polypeptide chain similar to the individual polypeptide chains found in hemoglobin. § The interior of Mb is composed of nonpolar amino acids. They are stabilized by hydrophobic interactions. § Polar, charged amino acids are located on the surface where they can form hydrogen bonds with water. § The heme binding group of Mb rests in a nonpolar crevice. § There are two polar His residues in the crevice (fig B). § The imidazole ring of one binds to the Fe2+ via a coordinate bond. § The other helps stabilize oxygen binding with the Fe2+. § The protein (globin) portion of Mb creates an environment that allows reversible binding of oxygen. § Oxidation of Fe2+ in Mb is rare.  Hemoglobin: Structure and Function (αβ)1 & (αβ)2 transports H+ and CO2 to the lungs carries O2 from the lungs Factors effecting O2 binding to Hb. 1- pO2 2- pH / pCO2 / (Bohr Effect) 3- 2,3-bisphosphoglycerate § Hemoglobin (Hb) is found only in red blood cells. § Hb A is the major form in adults. § It has a tetrameric structure The Hb tetramer may be viewed as two identical dimers (αβ)1 & (αβ)2. Those two α-and two β-chains held together by noncovalent (ionic and hydrogen bonds) interactions, which hold the two dimers together. Each subunit has stretches of α- helices and a heme- binding pocket. There are hydrophobic aminoacids in the interior of each chain as well on the surface so that interchain hydrophobic interaction is strong. § The function of Hb is more complex than Mb. Hb transports H+ and CO2 from tissues to the lungs. It carries O2 from the lungs to the tissues. § O2 binding is regulated by allosteric effectors.  Oxygen binding to Myoglobin and Hemoglobin Mb + O2 MbO2 * One heme chain-One O2 bound * Four heme chains-Four O2 bound § O2 binding shifts to the right or left as pO2 is increased or decreased. § Hb carries O2 to muscles. § The pO2 is lower in muscle than in blood & the affinity of Mb for O2 is greater than the affinity of Hb for O2. § Mb binds O2 as it is released by Hb. Mb then releases the O2 to muscle tissue in response to demand. § The sigmoidal shape of the Hb curve indicates that binding of O2 to the Hb subunits is cooperative, which means binding of O2 to one heme subunit increases the affinity of remaining subunit’s affinity for O2.  Carbon monoxide (CO) and Hemoglobin/Myoglobin The Carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+. § CO has similar size and shape to O2; it can fit to the same binding site. § CO binds heme over 20,000 times better than O2 because the carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+. § The hemoglobin’s protein pocket decreases affinity for CO, but it still binds about 250 times better than oxygen. § CO is highly toxic, as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation. § Treatment : 100% O2  Abnormal Hemoglobins Sulf-Hb: Forms due to high sulfur levels in blood. Sulfhemoglobinemia is a rare condition that can result from exposure to any substance containing a sulfur atom with the ability to bind to hemoglobin. Cases of sulfhemoglobinemia have been reported from ingestions of phenacetin, dapsone, and sulfonamides. (irreversible rxn) Carboxy-Hb: CO replaces O₂ and binds 220X tighter than O₂. Carboxyhemoglobin levels do not always correlate with the clinical severity, especially in patients with prolonged, low-level exposures could also result in high tissue concentrations of carbon monoxide (in smokers) Met-Hb: Contains oxidized Fe³⁺ (~2%) that cannot deliver O₂ to tissues. There are two types of methemoglobinemia: acquired and congenital. Benzocaine in babies or contaminated well water with excess nitrates can cause methemoglobinemia. Sometimes it is called baby blue syndrome due to cyanosis. (reversible with methylene blue infusion) § They are unable to carry and/or transport O₂ due to abnormal structure.  Minor Hemoglobins Fetal Hemoglobin (HbF)) HbA2 HbA1c Tetramer with two α and two Composed of two α and Two α and two β chains g chains two d globin chains -Glucose attached HbA1c levels are high Major hemoglobin found in Appears shortly before in patients with diabetes the fetus and newborn birth. mellitus Higher affinity for O2 than Constitutes ~2% of HbA undergoes non- HbA total Hb enzymatic Transfers O2 from glycosylation maternal to fetal Glycosylation circulation across depends on plasma placenta glucose levels Has been used for assessing blood glucose levels in diabetes patients (average of 3 mo.)  Anemia: Types of anemia: § Iron deficiency anemia. § Vitamin deficiency anemia. § Anemia of chronic disease § Aplastic anemia. § Anemias associated with bone marrow disease § Hemolytic anemia. § Sickle cell and other anemias. Anemia is a condition in which body does not have enough healthy red blood cells and/or Hb to carry adequate oxygen to the tissues. Different types of anemia Iron deficiency anemia. The most common type of anemia worldwide, caused by a shortage of iron in your body. Your bone marrow needs iron to make hemoglobin. Without adequate iron, your body can't produce enough hemoglobin for red blood cells. Vitamin deficiency anemia. Folate and vitamin B-12 are required to produce red blood cells. A diet lacking in these and other key nutrients can cause decreased red blood cell production. Some people may consume enough B-12, but their bodies aren't able to process the vitamin. This can lead to vitamin deficiency anemia, also known as pernicious anemia. Anemia of chronic disease. Certain diseases, such as cancer, AIDS, rheumatoid arthritis, Crohn's disease and other chronic inflammatory diseases which can interfere with the production of red blood cells. Aplastic anemia. This rare, life-threatening anemia occurs when your body doesn't produce enough red blood cells. Causes of aplastic anemia include infections, certain medicines, autoimmune diseases and exposure to toxic chemicals. Anemias associated with bone marrow disease. A variety of diseases, such as leukemia and myelofibrosis, can cause anemia by affecting blood production in your bone marrow. Hemolytic anemias. Develops when red blood cells are destroyed faster than bone marrow can replace them. Sickle cell anemia and Other anemias: Due to defective Hb.  Hemoglobinopathies: § Defects in these genes can produce abnormal hemoglobins and anemia, which are conditions termed "hemoglobinopathies". § Abnormal hemoglobins appear in one of three basic circumstances: 1. Structural defects in the hemoglobin molecule. Mutations can change a single amino acid building block in one or more the subunits. Occasionally, alteration of a single amino acid dramatically disturbs the behavior of the hemoglobin molecule 2. Diminished production of one of the two subunits of the hemoglobin molecule. Mutations that produce this condition are termed "thalassemias." Equal numbers of hemoglobin α and β chains are necessary for normal function. Hemoglobin chain imbalance damages and destroys red cells thereby producing anemia. 3. Abnormal associations of otherwise normal subunits. With severe α-thalassemia, the β-globin subunits begin to associate into groups of four (tetramers) due to lack of potential α-chain tetramers partners. These tetramers of β-globin subunits are functionally inactive and do not transport oxygen.  Hemoglobinopathies: Sickle Cell Disease Hydration Analgesics Antibiotics Transfusion RBC life span < 20 d Malaria point mutation at position 6 in the β-globin gene (Glu ® Val) Sickle Cell Disease (a.k.a. sickle cell anemia or HbS disease) § Most common blood disorder in the US (80,000 Americans) § 1 in 500 Americans of African heritage. § Homozygous recessive. § 1 in 12 are heterozygous (Have both HbA & HbS). These patients are considered to have sickle cell trait and usually do not exhibit symptoms. § Caused: a point mutation at position 6 in the β-globin gene (Glu ® Val). The mutant β- globin chains are designated βs, thus HbS is α2βS2. § Normal red blood cell lifespan is 120 days. It is < 20 days in HbS disease. § Sickle Cell disease is characterized by episodes of pain, chronic hemolytic anemia and increase susceptibility to infections. § Also, acute chest syndrome, stroke, splenic & renal dysfunction, bone changes (due to marrow hyperplasia). § At low oxygen tension, deoxyHbS forms a gel that subsequently assembles into a network of fibrous polymers that stiffen and distort the cell. § These misshapen red blood cells’ block capillaries, leading to localized anoxia, pain & eventually infarction near the blockage. § Treatment – hydration; analgesics; aggressive antibiotic therapy; – transfusion if high risk of fatal vessel occlusion. § Heterozygotes for the sickle cell gene are less susceptible to malaria (caused by Plasmodium falciparum). Short life span of red blood cells does not allow parasite to complete its life cycle  Hemoglobinopathies: Thalassemias Thallasemias § Normally, the rate of synthesis of the α- and β-globin chains of Hb are equal, hence α2β2 (HbA). § In the thalassemias, there is a heritable defect in the synthesis of either the α- or β-globin chain. § The thalassemias may be caused by a variety of mutations. – these include substitution or deletion of one or many nucleotide bases or entire gene deletions. § Thalassemias exist in which: – one of the globin chains is not produced (α0- or β0-thalassemia) or – some is synthesized at a reduced level (α+- or β+-thalassemia).  Hemoglobinopathies: Hemoglobin C Disease: Hemoglobin SC Disease: HbC crystals HbSC crystals Glu à Lys @ 6th position of β-chain Hemoglobin C disease: § Cooley's hemoglobinemia (HbC) is characterized by substitution of glutamate by lysine in the sixth position of β-chain. § HbC disease occurs mostly in African-Americans. § Both homozygous & heterozygous individuals of HbC disease are known. § It is characterized by mild hemolytic anemia. § No specific therapy is recommended. Hemoglobin SC Disease: § Both β-globin chains are abnormal, one exhibits the HbS mutation while the other exhibits the HbC mutation. Thus, they are doubly heterozygous (compound heterozygote). § The erythrocyte hemoglobin levels is higher (approaching normal) than in sickle cell disease. § Individuals with HbSC are often undiagnosed until they suffer a potentially fatal infarctive crisis. This is often precipitated by surgery or childbirth.  Membrane proteins A membrane protein is a protein molecule that is attached to, or associated with, the membrane of a cell or an organelle. Functions of membrane proteins: § Signal transduction: When the binding of the messenger to the receptor triggers a chain reaction involving other proteins, which relay the message to a molecule that performs a specific activity inside the cell. § Cell-cell recognition: The ability of a cell to distinguish one type of neighboring cell from another. § Intercellular joining: When membrane proteins of adjacent cells may be hooked together in various kinds of intercellular junctions. § Enzymatic activity: When a protein built into the membrane may be an enzyme with its active site exposed to substances in the adjacent solution. § Cell-cell recognition: When some glycoproteins (proteins bonded to short chains of sugars) serve as identification tags that are specifically recognized by other cells. § Attachment to the cytoskeleton and extracellular matrix: Elements of the cytoskeleton and the extracellular matrix may be anchored to membrane proteins, which help maintain cell shape and fix the location of certain membrane proteins. Others play a role in cell movement or bind adjacent cells together.  Fibrous Proteins Structural proteins or storage proteins that are typically inert and water-insoluble Special mechanical properties § Fibrous protein: forms long protein filaments, which are shaped like rods or wires, structural proteins or storage proteins that are typically inert and water-insoluble, occurs as an aggregate due to hydrophobic side chains that protrude from the molecule, e.g. collagen, keratin. § A fibrous protein exhibits special mechanical properties, resulting from its unique structure, which are obtained by combing specific amino acids into regular, secondary structural elements.  Collagen § Collagen is the most abundant protein in the human body. § A typical collagen molecule is a long, rigid structure in which three polypeptides(referred to as α-chains) are wound around one another in a rope like triple helix. § The collagen superfamily of proteins include more than twenty collagen types, as well as additional proteins that have collagen like properties. § The three polypeptide chains are held together by hydrogen bonds between the chains.  Collagen Structure –Gly–X–Y– X : proline and Y : hydroxyproline or hydroxylysine. Polytripeptide sequence can be represented as (–Gly–Pro–Hyp–)333. ü Increased interchain hydrogen bond formation Amino acid sequence: Collagen is rich in proline and glycine. Proline facilitates the formation of the helical conformation of each α chain because its ring structure causes “kinks” in the peptide chain. The presence of proline dictates that the helical conformation of the α chain cannot be an α helix. Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. Triple-helical structure: Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that places many of its amino acid side chains on the surface of the triple-helical molecule. This allows bond formation between the exposed R-groups of neighboring collagen monomers, resulting in their aggregation into long fibers. Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline and hydroxylysine, which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains. The hydroxylation is an example of posttranslational modification. Hydroxyproline is important in stabilizing the triple-helical structure of collagen by maximizing the interchain hydrogen bond formation.  Elastin Tropoelastin Proline Lysine Hydroxproline Hydroxlysine § Elastin is insoluble protein polymer. Tropoelastin secreted by the cell into the extracellular space interacts with the specific gycoprotein microfibrils, such as fibrilin. § Elastin is also rich in proline and lysine but contains only a little hydroxyproline and NO hydroxylysine. § Elastin fibrils have DESMOSINE cross links § In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue protein with rubber-like properties. § Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments. § They can be stretched to several times their normal length, but recoil to their original shape when the stretching force is relaxed.  Collagen related diseases: Osteogenesis imperfecta (OI) replacement of Gly with residues with bulky side chains resultant pro-α chains are unable to form the triple helix Osteogenesis imperfecta (OI): § It is also known as brittle bone syndrome, a heterogeneous group of inherited disorders distinguished by bones that easily bend and fracture. § Retarded wound healing and a rotated and twisted spine leading to a “humped- back” (kyphotic) appearance are common features of the disease. Type II is lethal. § Most patients with severe OI have mutations in type I collagen (in the gene for pro-α1 or pro-α2 chains). Mutations cause replacement of Gly with residues with bulky side chains. The resultant pro-α chains are unable to form the triple helix. § Type I OI (ostergenesis imperfecta tarda): Decreases production of α1 & α2 chains § Type II OI (ostergenesis imperfecta congenita)  Collagen related diseases: Ehlers-Danlos syndrome (EDS) ü a deficiency of collagen-processing enzymes (lysyl hydroxylase or procollagen peptidase) ü mutations in the amino acid sequences of collagen types I, III, or V Ehlers-Danlos syndrome (EDS): § It is a heterogeneous group of generalized connective tissue disorders that result from inheritable defects in the metabolism of fibrillar collagen molecules. § EDS can result from a deficiency of collagen-processing enzymes (e.g lysyl hydroxylase or procollagen peptidase), or from mutations in the amino acid sequences of collagen types I, III, or V. § The most clinically important mutations are found in the gene for type III collagen. § Patients with EDS show a defect in type 1 fibrils in the skin, leading to fragile, stretchy skin and lose joints. § Because collagen type III is an important component of the arteries, potentially lethal vascular problems.  Elastin related diseases: Marfan Syndrome (MFS) mutation in the fibrillin-1 protein Marfan Syndrome (MFS): § It is autosomal dominant connective tissue disorder affecting the microfibrils and elastin in connective tissue throughout the body. § Caused by a mutation in the fibrillin-1 protein, a glycoprotein that is secreted to extracellular matrix by fibroblasts. § Fibrillin is incorporated into insoluble microfibrils, that appear to provide a scaffold for deposition of elastin. § MFS is associated with pathological manifestations in the cardiovascular system (e.g., mitral valve prolapse, aortic aneurysm, and dissection), the musculoskeletal system (e.g., tall stature with disproportionately long extremities, joint hypermobility), and the eyes (e.g., subluxation of the lens of the eye). § 60 to 70 percent of people with Marfan Syndrome may have a dislocation of the lens of their eyes.  α1-antitrypsin and elastin degradation Role of α1-antitrypsin in elastin degradation: § α1-antitrypsin is a plasma protein which has an important physiological role of inhibiting neutrophil elastase. § A number of different mutations in the α1 antitrypsin gene are known to cause a deficiency of this protein, but one single purine base mutation, resulting in the substitution of lysine for glutamic acid is clinically the most widespread. § Smoking causes the oxidation and subsequent inactivation of methionine residue, thereby rendering the inhibitor powerless to neutralize elastase.