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Proteins

Adrianna Bialecka Biomolecular Sciences University Englisch 166 Karten

Proteins

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  1. 1What effect does water have on attractive forces?Weakens them
    e.g. Na and Cl apart (hydrated)
  2. 2What bonds does water form?Hydrogen bond
  3. 3What does the structure of ice look like?Each molecule of water can have max 4 hydrogen bonds with other water molecules
  4. 4How did simple organic molecules chemically evolve?Condensed to form more complex molecules or combined end to end as polymers of repeating units
  5. 5What functional group or linkage is -SH?Sulfhydryl group
  6. 6What functional group or linkage is -S-C=OThioester linkage
  7. 7What functional group or linkage is -S-S-?Disulfide linkage
  8. 8What functional group or linkage is =C=N-?Imino group
  9. 9What functional group or linkage is this?Phosphoryl groupWhat functional group or linkage is this?
  10. 10What functional group or linkage is this?Phosphoanhydride groupWhat functional group or linkage is this?
  11. 11What functional group or linkage is this?Phosphodiester linkageWhat functional group or linkage is this?
  12. 12What is Melanin?Produced by oxidation of the amino acid tyrosine followed by polymerizationWhat is Melanin?
  13. 13What is compartmentation in cells?Cell compartmentalization refers to the way organelles in eukaryotic cells live and work in separate areas within the cell in order to perform their specific functions more efficiently.
  14. 14Why is compartmentation efficient?->An enclosed system can maintain high local concentrations of components that would otherwise diffuse away
    ->More concentrated substances can react more readily, leading to increased efficiency in polymerization and other chemical reactions
  15. 15What are prokaryotes?organism that lacks a nucleus
  16. 16What are Eukaryotes?organism that contain membrane bound nucleus
  17. 17Why are viruses not classified as cells?lack the metabolic machinery to reproduce outside their host cell
  18. 18What does Amphipathic mean?Hydrophilic and hydrophobic
  19. 19What are micelle and bilayer in terms of membranes?What are micelle and bilayer in terms of membranes?
  20. 20What nucleic acid monomers bind to make polymers?What nucleic acid monomers bind to make polymers?
  21. 21Protein production diagramProtein production diagram
  22. 22How many amino acids are there?20
  23. 23Describe the structure of a typical amino acid?Have a primary amino group as a substituent of the alpha carbon atom (carbon next to the carboxylic group)
  24. 24How is a dipeptide formed?CO-NH bond is made with the elimination of a water molecules (H2 removed from amino and an oxygen from carboxylic acid)
    CONDENSATION REACTION    How is a dipeptide formed?
  25. 25How is a disulfide bond formed?Two cysteines join together (2 thiol groups are oxidised)
    OXIDATION REACTION    How is a disulfide bond formed?
  26. 26What does 50 residues long mean?50 amino acids long
  27. 27Which amino acid acts as a start codon?Methionine
  28. 28Can proteins have no AUG amino acid?Yes but only after processing the amino acid in post-translation modification part of the protein synthesis process
  29. 29What is a function of amino acids?Function as chemical messengers for communication between cells
    e.g. GABA, Dopamine, Histamine, Thyroxine
  30. 30What conformation do protein amino acids usually have?Trans conformation
  31. 31What is the primary structure?Amino acid sequence in a polypeptide chain
  32. 32What is the secondary structure?Alpha helix or beta sheet
  33. 33What is the tertiary structure?One complete protein chain
    e.g. Beta chain of hemoglobin
  34. 34What is the Quaternary structure?The four separate chains of protein assembled into a full protein
  35. 35What is an alpha helix?Right handed helical conformation has 3.6 residues per turn. A pitch of 5.4 angstroms- distance the helix rises along its axis per turn
  36. 36What is a beta sheet?Antiparallel beta sheets and parallel beta sheetsWhat is a beta sheet?
  37. 37What is the protein in jellyfish that fluoresces?GFP- Green Fluorescent Protein
    -conjugated double bond gives the protein its fluorescent properties    What is the protein in jellyfish that fluoresces?
  38. 38What does it mean when side chains are on the outside and not hiding in cytochrome?They are hydrophilic side chains
  39. 39What does it mean when side chains are hiding in cytochrome?Hydrophobic sides chains
  40. 40What does the Beta alpha beta motif look like?What does the Beta alpha beta motif look like?
  41. 41What does the beta hairpin motif look like?What does the beta hairpin motif look like?
  42. 42What does an alpha alpha motif look like?What does an alpha alpha motif look like?
  43. 43What does a greek key motif look like?What does a greek key motif look like?
  44. 44What is the N terminal of a protein?Start of the protein (primary amine group)
  45. 45What is the C terminal of a protein?End of a protein
  46. 46What shape do extended beta sheets often roll up to form?Barrels
  47. 47What do the two domains in the protein GAPDH do?-> N-terminal domain binds NAD+
    -> C terminal domain binds glyceraldehyde-3-phosphate
  48. 48What the difference between motifs and domains?Motifs are the super secondary structure of the protein and they perform similar biological functions while domains are a part of the tertiary structure of a protein and they exist and function independently from the protein chain
  49. 49What type of repeating structure does the fibrous protein keratin have?Alpha keratin is coiled coil
    -Exhibits 5.1 Angstrom spacing rather than the 5.4 Angstrom distance corresponding to the pitch of the alpha helix
    -Result of two a-keratin polypeptides each form an alpha helix twisting around each other to form a left handed coil
  50. 50What do two keratin polypeptides form?dimeric coiled coil
  51. 51What are protofilaments?Formed from two staggered rows of head to tail coiled coils
  52. 52What are protofibrils?dimerised protofilaments
    (two protofilaments)
  53. 53What are microfibrils?four protofibrils
  54. 54What is keratin rich in?Cysteines which forms disulfide bonds that crosslink adjacent polypeptide chains
  55. 55What does the structure of collagen look like?Triple helix - One third of its residues are Gly, 15-30% are Pro and another aa.
    Sometimes 4,5 and 3- hydroxyprolyl also occur in collagen but in smaller amounts. These residues are modified after the collagen peptides are synthesised.
    Pro residues are converted to hyp by prolyl hydroxylase which requires vitamin C -Scurvy = Vitamin C deficiency- Newly synthesised collagen cannot form fivers properly resulting in lesions
  56. 56What is the reaction pathway for cross-linking side chains in collagen?->Lysyl oxidase- catalysed oxidative deamination of Lys to form the aldehyde allysine
    ->Two allysines undergo aldol condensation to form allysine aldol
    ->This can react with His to form aldol histidine
    -> This in turn can react with 5-hydroxylisine to form an imine bond, crosslinking four side chains
  57. 57What is Myoglobin?Oxygen transport protein in the muscle
    -153 aa monomer
    -8 alpha helices
    -iron containing Haeme group
    - Major physiological role is to facilitate oxygen diffusion in muscle
  58. 58What is the structure of a Haeme group?Central Fe (II) atom is liganded to four N atoms of the Porphoryin ring. Pyrrole groups (4 of them). The Fe(II) is also liganded to a His side chain. This structure binds oxygen. An amino acid from different part of protein attached to iron (folding)
  59. 59Where is the Haeme complex in myoglobin?Wedged in a hydrophobic pocket between the E and F helices in myoglobin. Histidine E7 hydrogen bonds to the oxygen. Two hydrophobic side chain on the O2 binding site of the haeme (Val E11 and Phe CD1 help hold the haeme in place)
  60. 60What is the curve of Myoglobins oxygen binding?Hyperbolic, Myoglobin good at attacking O2 quickly
  61. 61What is Haemoglobin?A tetromer with two conformations. Responsible for oxygen transport in the blood from the lungs to the tissues
  62. 62What is the protein quaternary structure of haemoglobin?Tetramer- alpha 2 beta 2
  63. 63What is deoxyheamoglobin and heamoglobin?What is deoxyheamoglobin and heamoglobin?
  64. 64What causes the release of oxygen?low partial pressure
  65. 65What amino acids are hydrogen bonded to eachother in the T state?Asp G1, Tyr C7
  66. 66What amino acids are hydrogen bonded to eachother in the R state?Asn G4 and Asp G1
  67. 67What is Haemoglobins Oxygen binding curve?Sigmoidal
  68. 68What induces conformational changes in the subunits?Ligand binding
  69. 69What is the role of Haemoglobin and Myoglobin in O2 and CO2 transport?O2 is inhaled at the lungs at high pO2 binds to hemoglobin in blood. Transported to respiring tissue where pO2 is low- O2 dissociates from Hb diffuses into the tissues. Oxidizes metabolic fuels to CO2 and H2O. In rapidly respiring muscle O2 first binds myoglobin increasing the rate at which O2 diffuses from the capillaries by effectively increasing its solubility
  70. 70What produces antibodies?B-cells (lymphocytes that mature in the bone marrow.
  71. 71What is the most abundant antibody?IgG
  72. 72How to remember antibodies?MADGE
  73. 73What does the X-ray structure of immunoglobulin G (ribbon form) look like?4 blue and 4 yellow immunoglobin folds and 2 pink immunoglobulin folds
  74. 74What does the immunoglobulin fold look like?The polypeptide chain is folded into a sandwich of 3 and 4 stranded antiparallel Beta sheets
  75. 75What does the IgG consist of?4 subunits (4 polypeptide chains)
    -2 heavy polypeptide chains
    -2 light polypeptide chains
    12 immunoglobulin folds- antiparallel beta sheets
  76. 76Diagram of a IgGDiagram of a IgG
  77. 77Where does glycolisation occur in immunoglobulins?at CHO groups
  78. 78What is the function of the hinge region?Gives flexibility
  79. 79What does PAGE stand for?Polyacrylamide Gel Electrophoresis
  80. 80What does Fc stand for?Crystallizing fragment
  81. 81What does FAB stand for?Antigen-binding fragment
  82. 82What does CDR stand for?Complementarity-determining regions
    -each containing 3 hypervariable loops that bind the antigen
  83. 83What is an epitope?Specific part of an antigen recognised by an antibody
  84. 84Where is the antigen-binding site located?At the tip of Fab Fragment in a crevice between VL and VH domains
  85. 85What does it mean when it is said that IgG has divalent binding?Can bind two identical antigens simultaneously
  86. 86Why do antibodies cross-link to form a lattice?Speeds up removal of antigen and triggers B-cell proliferation
  87. 87How are monoclonal antibodies generated?Lymphocytes will not grow continuously without being immortalized. They immortalize lymphocytes by fusing with myeloma cells (hybridoma cells). Only the fused hybridoma cells can grow in a special selective medium
  88. 88What are the uses of monoclonal antibodies in the laboratory?- protein purification and identification in the lab
    - Identification of diseases
    - Therapeutic agents
  89. 89What is one example of an application for antibodies for labelling/tagging proteins of interest?ELISA
    Enzyme linked immunosorbent assay
  90. 90Why do we label/tag proteins of interest?-to determine if a particular protein is present in a mixture
    -to quantify the amount of the protein present
    -to separate a specific protein from a mixture
  91. 91How does ELISA work?1. Immobilize first antibody on solid support
    2. Incubate with protein-containing sample
    3. Add a second antibody that is covalently linked to an assayable enzyme
    4. Wash and assay enzyme activity. Amount of substrate converted to product indicates amount of protein present
  92. 92What can SARS-CoV-2 serological testing not do?Doesn't detect active infection
    (uses patients blood)
  93. 93How do antigen tests work?There is a control antibody and an antibody against antigen on the test. When sample is applied a positive result would occur when the antigen sticks to both control and antibody against antigen.
    A negative results would be no antigens stuck to either
  94. 94What are biopharmaceuticals?-recombinant proteins, including recombinant antibodies (mAbs). We can recreate in the lab
    -nucleic acid-based products
    -genetically engineered cell-based products
  95. 95Example of a monoclonal antibody used to treat chronic diseaseHumira- IgG monoclonal antibody used to treat inflammatory conditions such as rheumatoid arthritis and Crohn's disease.
    Binds and neutralizes TNF-alpha, a pro-inflammatory cytokine
  96. 96Example of a monoclonal antibody used to treat cancerHerceptin- Monoclonal antibody that binds and blocks HER2 receptor signaling
    Trodelvy- Antibody-drug Conjugate (drug is attached to antibody), Used to treat Triple Negative Breast Cancer. Mechanism of action: binds Trop-2 Receptors. Cell internalises the ADC and the drug, SN-38 (a topoisomerase inhibitor) is released and exerts its cytotoxic effects
  97. 97Example of monoclonal antibody used to treat infectious diseaseRonapreve- Contains two monoclonal antibodies that target two different parts of the spike protein on Sars-CoV-2. Block the spike protein attaching to ACE2 receptors on the cells of the respiratory tract
  98. 98What two proteins are involved in skeletal muscle contraction?Myosin and actin
  99. 99What do Muscle fibres look like?they look striated. Very long, multinucleated cells. Produced by cell fusion. Consist of myofibrils
  100. 100What do Myofibrils contain?Thick filaments and thin filaments. Are surrounded by flattened vesicles containing Ca 2+ called sarcoplasmic reticula (nerve impulses cause Ca 2+ release)
  101. 101What is a Fascicle?Bundle of muscle fibers
  102. 102What is the I band of the myofibril?contains only thin filaments
  103. 103What is the A band of the myofibril?contains only thick filaments in the H zone. Darker outer segments contain overlapping thick and thin filaments
  104. 104What is the sarcomere?the functional unit of contraction
  105. 105What happens during myofibril contraction?During contraction, the length of each filament does not change. But the sarcomere reduces in the length due to overlapping/sliding of the filaments
  106. 106Where are the key proteins involved in Muscle contraction found?Myosin (thick filaments)
    Actin (thin filaments)
    Tropomyosin and troponin (in thin filaments, inhibitory role)
  107. 107What is the protein structure of Myosin?6 polypeptide chains
    -2 identical heavy chains: each with an N-terminal globular head (myosin head) and an alpha helical tail
    -Each heavy chain associates with two kinds of light chains
    ->Essential light chain (ELC)
    ->Regulatory light chain (RLC)
    - The two alpha helical tails wind around each other to form a 1600 Angstrom long parallel coiled coil
  108. 108Myosin structureMyosin structure
  109. 109What is the function of the myosin head?ATPase and has an ATP-binding site
  110. 110What is the structure of a thick filament?Several hundred myosin molecules form a staggered array with their globular heads pointing away from the filament
  111. 111What is the structure of G-Actin Structure?Most abundant cytosolic protein in eukaryotes- G-actin is the monomeric form
    (375 residues). Four protein domains, Binding sites for ATP in the cleft
  112. 112What is F-action?300-400 globular G-actin monomers polymerize to form a fibrous polymer of actin known as F-actin. F-actin polymers form double-stranded helices
  113. 113What is the Myosin-Actin interaction?In a myofibril every actin monomer has the potential to bind one myosin head.
  114. 114What is the mechanism of force generation in Muscle?1. ATP binds to myosin heads; actin binding site opens; myosin head releases actin. ATP attaches -> change in conformation of myosin head
    2. Active site closes and ATP is hydrolyzed, causing cocking of myosin head (Perpendicular)
    3. Myosin head binds weakly to the actin
    4. Pi is released, resulting in strong binding of myosin head to actin
    5. Power stroke: A conformational shift that sweeps the attached thin filament towards the M disc
    6. ADP is released
    Undirectional process
    Contraction cycles must be repeated many times to contract muscle
  115. 115What is Tropomyosin?dimer of alpha helical subunits that form a coiled coil. The coils join to form cables that runs the length of the F-actin filaments
  116. 116What is troponin?Consists of 3 subunits
    -> Troponin C (binds Ca 2+)
    -> Troponin I (binds actin, prevents binding of the myosin head)
    -> Troponin T (binds tropomyosin)
  117. 117What does the tropomyosin-troponin complex do in a relaxed muscle?Blocks the myosin-binding sites on actin
  118. 118How is muscle contraction triggered?Nerve impulse-> Ca 2+ released from sarcoplasmic reticulum -> intracellular Ca 2+ increases. Ca 2+ binds troponin C which triggers conformational change in tropomyosin-troponin complex to remove the inhibition by troponin I and expose the myosin binding site of actin
  119. 119Why do muscles get thicker after exercise?Increased muscle fiber diameter known as Skeletal Muscle Hypertrophy. More myofibrils are added to each muscle fiber to increase muscle mass
  120. 120What happens when you damage a muscle?New muscle fiber cells are generated
  121. 121What are satellite cells?Muscle stem cells, located between the sarcolemma and basement membrane of muscle fibers.
    Involved in normal growth of muscle, muscle plasticity and regeneration after injury/disease
    Main source of new myonuclei in postnatal skeletal muscle
  122. 122How do Satellite cells fix damaged muscle?Differentiate and fuse with each other to form new muscle fibers or differentiate and fuse with existing fibers to supply additional myonuclei
  123. 123How does smooth muscle contract ?Elongated, spindle-shaped cells with a single centrally located nucleus. Contraction is involuntary, control by the autonomic nervous system (ANS).
    Smooth is not Straited (filaments not arranged into sacromeres
    Ca 2+ from extracellular fluid binds calmodulin (no troponin c)
  124. 124What do Actin microfilaments play a role in?Changes in cell shape
    Cell division
    Cellular locomotion
    Endocytosis
    Organelle transport
  125. 125In a crawling macrophage which edge does what?Leading edge in the process of extending
    Trailing edge is gradually being pulled
  126. 126How do cells move?Microfilament treadmilling.
    Actin monomers continually add to the positive end of the filaments but dissociate at the same rate from the negative end. Exerts force of plasma membrane.
    Allows cytoplasm to move in one direction
    (ACTIN-MEDIATED CELL LOCOMOTION IS DRIVEN BY ATP HYDROLYSIS)
  127. 127What are some examples of diseases caused by a dysfunctional protein?-Sickle cell anaemia
    -Alzheimers disease
    -Prion disease
    -Cystic fibrosis
  128. 128What is the cooperative interaction between haemoglobin and oxygen?O2 binding leads to a conformational change in quaternary structure of haemoglobin -> Increases its affinity for O2 at the other O2 binding sites
  129. 129What is the most common way mutant haemoglobin forms?Single amino acid substitution
    Mutations may
    -> Destabilise haemoglobins tertiary/quaternary structure
    -> Alter haemoglobin's O2-binding affinity
    -> Decrease cooperative interactions
  130. 130What is methemoglobin formation?Iron in ferric state = no affinity for O2
  131. 131What is Sickle cell anaemia?A single amino acid change in the beta subunits of hemoglobin results in Sickle Cell Anaemia. It contains a valine rather than a glutamic acid at the sixth position of each beta subunit (E6V). The valine 6 binds to a hydrophobic pocket on the beta subunit of a neighboring deoxyhemoglobin tetramer
  132. 132What is the structure of Deoxyhemoglobin 5 fibers?Attachment of Val 6 to a hydrophobic pocket on an adjacent beta chain, allows haemoglobin tetramers to form linear polymers. Aggregates of 14 strands that wind round each other form fibers
  133. 133What is the problem with sickle-shaped erythrocytes?Blood flow to tissues can be blocked resulting in tissue death
  134. 134What are the treatments for sickle cell anaemia?-Bone marrow transplant
    -Pain medication
    -Infection prevention
    -Hydration
    -Hydroxyurea (increases expression of foetal haemoglobin -contains y gamma instead of beta subunits)
    -Casgevy- cell based gene therapy
    -Lyfgenia- cell based gene therapy
  135. 135What is alzheimer's disease?By brain tissue that contains abundant amyloid beta plaques surrounded by dead and dying neurons. Extracellular depositions of normally soluble proteins form insoluble aggregates known as amyloid plaques. (indestructible)
  136. 136How do amyloids form?Amyloid beta precursor protein that is cleaved by alpha and gamma secretase to release to Amyloid beta peptide. Amyloid beta likes to dimerise, trimerise, ...oligmerise
  137. 137What are the strategies for targeting neurotoxic amyloid Beta-protein oligomers?Formation of oligomers and then amyloid fibrils may be decreased or prevented by?
    - Inhibition of alpha beta protein production
    - Stimulation of alpha beta protein degradation
    -Small molecules can bind to and stabilize alpha beta protein monomer (may prevent oligomerization and allow for the natural removal of monomers by the brain's degradative machinery)
    -Agents capable of disrupting pre-formed oligomers should reduce the concentration of toxic oligomers
    -Small molecule or antibodies capable of binding to a variety of alpha beta protein assemblies could neutralize both small and large alpha beta protein assemblies and facilitate their elimination
  138. 138What are some treatments for Alzheimer's Disease?Traditional treatments for Alzheimer's are largely symptomatic treatments
    -Cholinesterase inhibitors: donepezil, galantamine and rivastigmine
    -N-methyl-D-aspartate antagonist; memantine
    - Recently approved mAbs directly target the plaques (Lecanemab and Donanemab)
  139. 139Mad cow disease is an example of a what disease?Prion disease
  140. 140Examples of prion diseases-Scrapie (in sheep)
    -Bovine Spongiform Encephalopathy (BSE)-Mad Cow disease
    -Creutzgeldt-Jakob Disease (CJD)
    (all affect CNS)
  141. 141What is the Transmissible Spongiform Encephalopathy disease?Neurons develop large vacuoles giving brains spongy appearance. Infectious disease (infectious agent is a protein called a prion which is a particle that lacks nucleic acid). Prion proteins aggregate as clusters of rod like particles that closely resemble amyloid plaques and lead to neuronal degradation
  142. 142What are the key differences in prion protein conformations?Cellular PrPc is...
    -soluble
    -normally present in brain
    -function unknown
    -Extensive regions of alpha-helix
    -Very little beta-sheet
    Diseases PrPsc is....
    -Insoluble
    -Less alpha-helix regions
    -Many stacked beta-sheets
    -Aggregates
    -Captures and converts PrPc
  143. 143What are the treatments for Prion Diseases?-no cure
    -symptoms treated with pain relief and anti-depressants
    -Early stage of development= antibodies to PrPc
  144. 144What is Cystic Fibrosis?-Characterized by Chronic lung infections and lung damage
    - >1000 mutations but 70 percent are associated with defective Cl- transporter
  145. 145What is the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)?-ABC transporter = needs ATP to open
    -Normally allows Cl- ions to flow our of the cell following their concentration gradient. Cl- flux is accompanied by Na+ and water
    - Binding of two ATP molecules appears to open the channel . Hydrolysis of one ATP closes the channel
    - Maintains proper fluidity of the mucus in the respiratory tract, pancrease, sweat glands
  146. 146What is the delta F508 in Cystic Fibrosis?Specific mutations within the gene for the protein CFTR. The mutation if a deletion of a single codon for the amino acid phenylalanine (F). Results in an abnormal CFTR protein that cannot fold properly. It does not escape the endoplasmic reticulum for further processing.
  147. 147What does the mutation delta F508 cause?Affects chloride ion channel function leading to dysregulation of epithelial fluid transport in the lung, pancreas and other organs, resulting in cystic fibrosis. Complications include thickened mucus in the lungs with frequent respiratory infections, and pancreatic insufficiency giving rise to malnutrition and diabetes
  148. 148What are the treatments for Cystic Fibrosis?-Antibiotics to prevent and treat chest infections
    -Mucus thinners
    -Medicines to widen the airways and reduce inflammation
    -Physiotherapy to help clear mucus from the lungs
    -Transplant
    -CFTR modulators (Trikafta)
  149. 149What method of protein analysis is used to measure concentration?>U.V. Spectrophotometry
    -Aromatic side chains of Tryptophan, Tyrosine and Phenylalanine absorb light in the U.V. spectrum
    -These then emit light at a new wavelength
    -Inherent fluorescence of proteins
    - Absorbance coefficient of a protein is dependent of the aromatic side chain content
    >ELISA
  150. 150What methods of protein analysis are used to separate protein depending on characteristics?Chromatography and Polyacrylamide gel electrophoresis
    Solubility= Salting out
    Ionic Charge= Ion exchange chromatography, Electrophoresis and Isoelectric focusing
    Polarity= Hydrophobic interaction chromatography
    Size= Gel filtration chromatography
    SDS-PAGE
    Binding Specificity= Affinity Chromatography
  151. 151How are proteins separated by centrifuge in a salt gradient?A salt e.g. ammonium sulphate is added to a solution of macromolecules to a concentration just below the precipitation point of the protein of interest
    After centrifugation, the unwanted precipitated proteins are discarded and more salt is added to the supernatant to a concentration sufficient to precipitate the proteins of interest.
    After second centrifugation, the desired protein is recovered as a precipitate and the supernatant is discarded.
    This salting out is based on a competition between the added salt ions and the dissolved solute for molecules of solvent
  152. 152How are proteins separated using gel filtration chromatography to separate proteins based on their molecular weight?Larger proteins elute off before the smaller proteins. The agarose beads (gel) in the stationary phase are porous with small pores and channels permeating the beads. Larger proteins do no enter the beads through these pores while smaller ones do.
  153. 153What are the two types of ion exchange chromatography?Anion-exchange- column resin is positively charged attracting negatively charge proteins
    Cation-exchange- column resin is negatively charged attracting positively charge proteins
  154. 154How does ion exchange chromatography work to separate ions and polar molecules based on their affinity to the ion exchanger?Proteins bind to moieties which are oppositely charged by forming ionic bonds to the insoluble stationary phase. Cationic stationary phase is used to separate cations. Bound molecules eluted and collected by running higher concentration of salt through the column or changing pH of the column
  155. 155How does affinity chromatography work to separate proteins based on the binding affinity for the desired protein to another protein on the column bead (affinity ligand)?A ligand is immobilized by covalently binding it to the chromatographic matrix. A mixture of macromolecules is added to the column. Only certain molecules specifically bind to the ligand. The other components are washed through the column
  156. 156How are proteins separated according to molecular weight using Polyacrylamide Gel Electrophoresis?Samples are mixed in a sample bugger containg sodium dodecyl sulfate which denatures proteins and confers a negative charge, and DTT/ Beta mercaptoethanol: reducing agents (breaks disulphide bonds)
    Sample is boiled for 5 mins.
    Samples are loaded into wells in a vertical polyacrylamide gel
    A current is passed through the gel, larger proteins are retarded towards the top of the gel
  157. 157How are proteins separated by charge through two dimensional gel electrophoresis and isoelectric focusing IEF?The isoelectric point is the pH at which a particular molecules carries no net electrical charge. At a proteins pl it is immobile in an electrical field. If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode- each protein will migrate to its position of the pH gradient corresponding to its pl. Valuable tool in proteomics
  158. 158How is the proteins structure obtained using mass spectrometry?Protein of interest is digested into peptide fragments with a protease e.g. trypsin- then dissolved in a volatile solvent. Dry N2 gas promotes the evaporation of the solvent from charged droplets containing the protein of interest- leaves gas phase ions whose charge is due to protonation of Arg and Lys residues. The mass spectrophotometer then determines the mass to charge ratio of these ions.
    The resulting mass spectrum consists of a series of peaks corresponding to ions that differ by a single ionic charge and the mass of one proton
  159. 159What is the Tandem Mass spectrometry? MS/MSPeptides are separated by the first MS and one peptide is sent through a collision cell where it collides with helium atoms. This breaks the peptide into further fragments which are directed into a second MS that determines the m/z values of these smaller fragments
  160. 160How does X-ray crystallography work to give ribbon images of proteins?1. Precipitation of a pure preparation of the protein
    2. Crystal formation- solid crystals of pure molecules
    3. X-Ray diffraction through the crystal- reveals a pattern
    4. Capture of the diffraction pattern and rendering by computer software into mathematical co-ordinates
    5. Deposition of the Crystal Structure into database
  161. 161How does Cryogenic Electron Microscopy (cryo-EM) work?Produces 3D images in atomic details (can show molecule dynamics)
    Purified sample is added to a grid with tiny holes. Samples in the grid are rapidly frozen in liquid ethane (vitrification) which stops the movement of cellular components so the electron beam can get clear images, protects the molecules from the electron beams and prevents crystal formation. An electron beam travels through the sample and casts 2D shadows or projections of the molecules onto a detector. Image processing
  162. 162What is the function of E7 histidine?Act as a gate with an open or closed conformation controlling access to the active site
  163. 163What is the function of Val E11?orient bound oxygen toward the epsilon amino nitrogen of His-E7 for more efficient hydrogen bonding
  164. 164What is the function of His F8?Contribute to the coordinated binding of Fe in heme
  165. 165What is the function of Phe CD1?Stabilizes the haem by direct contact
  166. 166—

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University Biomolecular Sciences Karteikarten am Proteins, erstellt von Adrianna Bialecka am 16/09/2024.
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