Midterm 2 Flashcards
5 Major Function Protein Classes
Metabolism, Structure, Transport, Cell Signaling, and Genomic Caretakers
(not totally inclusive and some fit into more than one group)
What are Metabolic Enzymes?
reaction catalyst that control metabolic flux
Facts about metabolic enzymes
●increases rate of product formation by lowering activation energy
●DOES NOT CHANGE DELTA G
●Responsible for the synthesis and degradation of macromolecules
●Amino acid side chains specify shape and chemical environment of enzyme active site (helps lower activation)
●most are part of multisubunit protein complexes (quarternary)
●names based on reactant and describe mechanism of reaction
What are Structual Proteins?
Most abundant proteins in living organisms and they function as architectural framework for individual cells, tissues, and organs
Structural protein facts
●Remember collagen
●maintain integrity of cell structures and promote changes in cell shape
●Cytoskeleton protein: responsible for cell shape, cell migration, and cell signaling
Examples of Cytoskeleton Proteins
Actin, Tubulin, and Collagen
Actin
●Abundant cytoskeletal protein in animal cells
●Found in muscles
● subunits self assemble from actin monomers to polymers called thin filaments
Function of thin filaments
●molecular cables controlling cell shape and cell migration
●also used in muscle contraction
Tubulin
●in animal cells
● self assemble from tubulin monomers to long polymers called microtubules
Function of microtubules
●act as “road” for movement of organelles and chromosomes during cell division
●pushes them together and pulls them apart (Mitotic checkpoint)
Collagen
●fibrous protein
●3 intertwines left-hand helices
●repeating Gly-Pro-4HyP tripeptide
●left handed helix with 3AA/turn
●Stabilized by interstrand hydrophobic interactions (make tight right hand triple helix)
What are Transport Proteins?
membrane-spanning proteins that transport polar or charged molecules in and out of cell
●generally all transmembrane proteins are transport proteins
What are the two classes of transport proteins?
Passive and Active
Passive Transport Proteins
●Do not require energy to transport molecules across membrane
●in response to chemical gradients (high to low concentrations)
●examples: porins and ion channels
●controlled by amino acids in transport
●static!!
Active Transport Proteins
●Require energy to induce conformational change in protein to open or close gated channel
●pump small molecules or ions against concentration gradient
●Energy comes from ATP hydrolysis or Ionic gradient
●Example: Ca2+ ATPase transporter protein
●not static
What are Cell Signaling Proteins?
important for communication and includes receptors
What are Receptors?
●proteins that function for communication
● most important drug targets
●control and communication primarily come from brain and spinal column
Cell-Cell Communication with neurons
●brain sends messages as electrical pulse, travels down nerve cell (neuron) toward target
●Neurons don’t directly touch target, message carried across gap by neurotransmitters (always outside the cell)
●Binds to specific receptor on the cell membrane of target, leads to cascade of secondary effects (signal transduction cascade)
●Causes flow of ions across membrane or switches on and off enzymes inside target cells
Cell-Cell communication with hormones
●Chemical messenger, travel in blood, travel farther, travel bilayer (they are non polar)
●Binds and causes conformational change to switch on receptor molecule and message is received
What are the three different types of membrane-bound receptors?
ion channel receptors, G-protein-coupled receptors, and Kinase-linked receptors
Ion-Channel Receptors
●Complexes made of 5 protein subunits that transverse (transmembrane protein) the cell membrane (quaternary structure)
●hydrophilic tunnel (outside nonpolar, inside polar)
●receptor is part of 1 of the protein subunits
●uses regular nonpolar AA’s because it is small size and linear
●neurotransmitter to ion channel= fast response
G-Protein-Coupled Receptors
●Largest class of receptors, biggest drug target
●Globular protein with 7 transmembrane regions (hydrophobic and helical in shape), known as 7-TM receptors
●Examples: adrenergic receptors (adrenaline)
●Activated by hormones and slow-acting neurotransmitters
●GCPRs activate G-protein (signaling protein) which will activate/deactivate membrane bound enzymes or trigger production of secondary messengers
Angiotensin II Receptor
●Example of GPCR
●Angiotensinogen -> Angiotensin I (decapeptide) -> Angiotensin II (tetrapeptide)
●Messager binds to receptor, activates Angiotensin II by opening site of receptor for G-protein to bind, causes signal transduction that lowers BP
Kinase-Linked Receptors (KLRs)
●Phosphorylation
●Can be Tyr, Ser or Thr
● enzyme and receptor
●Reversibly phosphorylates proteins at Ser and Thr amino acid residues on downstream target proteins in response to upstream receptor activation signals
●critical role in regulating cell differentiation, proliferation, survival, metabolism, and migration
●important for anticancer
●activated by large number of hormones, growth factors, and cytokines
●Loss of function=developmental defects or hormone resistance
●overexpress= cancer (more receptors= more responses)
Domains of KLRs
●Ligand binding domain outside cell
●single membrane-spanning domain
●tyrosine kinase domain inside cell
Examples of KLRs
Mitogen-activated protein kinase, protein kinase A, insulin receptor, phosphoinositide-3 kinase
What is the binding result of KLRs
●receptor dimerization with adjacent receptor, causes tyrosine kinase domain to become active by conformational change when they come together
●Autophosphorylation: receptors phosphorylate each other on multiple tyrosine/ser/thr sites (need ATP to do it)(enzyme is not active until dimerization happens)
●Can then bind to other proteins (SH2 proteins) that specifically recognize the phosphorylated tyrosines
What are Genomic Caretaker Proteins?
●they maintain the integrity and accessibility of genomic information
●Important for repairing mutations in DNA reproductive cells (inherited by offspring)
●Includes DNA replication, repair, and recombination proteins (DNA polymerase, DNA ligase, topoisomerase, DNA primase, and RNA polymerase)
What is DNA ligase
●Enzyme that joins DNA strands together by forming phosphodiester bond
●Joins Okazaki fragments together during replication
●Used in DNA repair pathways
What is DNA primase?
Creates RNA primer for the polymerase to bind to
What does topoisomerase do?
●unwinds DNA
●relieves supercoil by cutting in 1 or 2 places
Ivosidenib
●metabolism drug target
●isocitrate dehydrogenase-1 inhibitor
●anticancer drug
●first in class drug
Methotrexate
●metabolism drug target
● inhibits enzymes responsible for nucleotide synthesis (this prevents cell division)
●anticancer
Fluorouracil
●metabolism drug target
●inhibits thymidylate synthase
●anticancer
What protein class is the most targeted by drugs?
Cell Signaling
What is tubulin a huge target for?
●Cancer
●drugs can stop tubulin extension or messes w/ attachment (leads to apoptosis at mitotic checkpoint
Vinca Alkaloids
●Structural drug target
●anticancer
●second largest
●VBL and VCN
Taxel
●Paclitaxel and docetaxel
●Structural drug target
●anticancer
Eribulin
●Structural drug target
●Natural produce is halichondrin B
●Smaller and more soluble
●Anticancer
Digitoxin
●Transport drug target
●ATPase inhibitor
●used to treat congestive heart failure (makes heart contract more)
TKR
●Cell Signaling drug target
●Anticancer
●Examples of KLR
What are examples of drug-targeting GPCRs?
histamine receptor blockers (allergy), opioid agonists, beta-blockers (high blood pressure), and angiotensin receptor blockers
Cyclosporine
●Cell signaling drug target
●immunosuppressant (for organ transplant)
●Calcineurin inhibitor (GPCR receptor)
Prazosin
●Cell Signaling drug target
●treats hypertension
●alpha-adrenergic blocker
●GPCR example
Vasopression
●Cell Signaling drug target
●BP medication
●targets multiple GPCRs
What are Genomic Caretaker proteins important drug targets for and what do they disrupt?
●important for anticancer
●Transcription/Translation Disruptors
Etoposide
●Genomic Caretaker drug target
● natural protein is podophyllotoxin
●Disrupts TOPII
●anticancer
Doxorubicin
●Genomic Caretaker drug target
●anticancer
●Disrupts TOPII
Camptothecin
●Genomic Caretaker drug target
●anticancer
●Topotecan and Irinotecan
●Disrupts TOPI
What happens is TOPII or TOPI are not functioning?
DNA damage accumulation that leads to apoptosis
DNA Repair Pathway Assassins
●Trabectedin: binds to minor groove (not reversible) -> DNA replication or transcription halted -> apoptosis
●first tries to repair with DNA repair
pathways -> proteins involved in DNA
repair are inhibited because it binds to
part of the molecule -> apoptosis
●multiple mechanisms of action
●PARP inhibitors (Olaparib): treat gBRCAm metastatic breast cancer
●CDK inhibitors (Ibrance): cancer therapy, treat HR-positive and HER2-negative breast cancer
Hemoglobin
●transport oxygen from lungs through circulatory system
●major protein in blood cells
Myoglobin
●storage depot for oxygen (saves it)
●concentrated in muscles (stationary in muscles)
What are structural similarities for hemoglobin and myoglobin?
●globin fold: eight alpha helices (similar to 4 helix bundle)
●bind oxygen reversibly to Fe2+ in porphyrin ring tightly bound to protein
Heme
●Fe2+ porphyrin complex
●prosthetic group: non-amino acid portion of certain protein molecules
●necessary since no amino acids side chains can reversibly bind oxygen
What is porphyrin?
group of heterocyclic, macrocyclic, organic compounds, composed of four modified pyrrole subunits
Heme in Myoglobin
single polypeptide chain with one heme group
Heme in Hemoglobin
●four polypeptides (two identical alpha and two identical beta subunits) with four heme groups
●considered a dimer of heterodimer (a1b1, a2b2) (functional significance for subunit interactions)
What is a ligand?
ions or neutral molecules that bind to a central metal atom or ion in a larger molecule (protein)
Ligand-Protein Interactions
●Ligand binding is reversible (noncovalent interactions)
●binding induces or stabilizes structural conformations in target proteins (alter protein affinity for ligand)
●Effector molecules can alter equilibrium between ligand-bound and ligand-free proteins (increase and decrease affinity)
Ka
●association constant
●P+L -> PL
Kd
●dissociation constant
●PL -> P+L
●larger Kd= lower affinity because larger products (P and L do not want to be together) and smaller amount of reactants
Fractional Saturation
●θ=fraction of protein binding sites that are occupied
●= Occupied binding sites/ Total binding sites
●can rearrange to L/L+Kd
Fraction Saturation graph facts
●ligand concentration=Kd, then θ=0.5
●θ vs [L] makes a hyperbolic curve
●Simple protein-ligand interaction
●Kd can be determined by concentration of ligand at 0.5
●think amount of ligand needed to saturate 50% of proteins
●higher affinity=less concentration needed to saturate 0.5
●IF graph does not plateau at 100, you need to do Kd based on half of where it plateaus
What can we learn from Kd
●what factors influence protein’s interaction with ligand to generate stronger or weaker binding
●which ligands are physiologically important
●the conditions that affect protein’s function
Fraction Saturation for Myoglobin
●θ vs pO2
● hyperbolic, simple
●more active= release O2, saturation drops
●rest= higher saturation, readily for muscle activity because O2 is used to form ATP
Fraction Saturation for Hemoglobin
●θ vs pO2
●sigmoidal curve, not simple
●sharper decrease in saturation at low oxygen
●Saturation gets lower away from lungs because it gives O2 to all the cells that need it
●indicates positive cooperative binding
Cooperative binding
●binding of the first ligand to the protein complex facilitates the binding of additional ligands on same protein
● need quaternary structure
●Myoglobin can not do cooperative binding because it only has one heme (1 subunit) (no 4 structure)
What are the 6 coordination bonds to the Fe2+ in the heme group?
●4 planar nitrogen
●proximal histidine: directly coordinates to Fe2+ (covalent)
●distal histidine: hydrogen bonds to O2, stabilize interaction with heme group
Deoxyhemoglobin
no oxygen in heme, no longer planar, causes pucker
Oxyhemoglobin
●O2 binding causes the Fe2+ to be pulled into plane of porphyrin ring (proximal moved with it)
● moves F8 Histidine and backbone (induces conformational change in entire molecule)
Cooperative Binding with small movements
●small movements in F helix from O2 binding causes entire protein complex to change noncovalent interactions at interface of alpha and beta subunits
●small structural changes in one region -> large structural changes in entire hemoglobin molecule
●alter O2 affinity in all 4 globin subunits
T (tense) State
●Oxygen is unbound
●deoxyhemoglobin
●larger hole in center
R (relaxed) State
●Oxygen is bound
●oxyhemoglobin
●aB dimer rotated 15 degrees from the other
●fourth O2 binds with 100x more affinity than first O2 does (different affinity from T)
Concerted Model
●Protein exist in either T or R state (exist in equilibrium)
●T favored when little for no ligand is bound
●Ligand binding to single subunit in tetrameric protein complex helps stabilize R state (increases population of R state)
●Ligand bind to T or R, but only high affinity for subunits in R state
●No mixture in hemoglobin, only T or R, whole molecule changes at once
Sequential Model
●ligand binding of one subunit of tetrameric protein induces conformational change so nearby subunits are more likely to bind ligand and shift to R state
●can be mixture of T and R in one molecule of hemoglobin
Allosteric Effectors
●Don’t need cooperative binding
●describes ability of biological molecules to transmit effects of binding spatially through the protein to other sites
●Molecules that alter structural conformation to favor one state over another
Hemoglobin Allosteric Effectors
●Oxygen (positive: encourage T -> R)
●2,3-biphosphoglycerate (negative: encourage R ->T):
●over negative effectors: CO2, H+
H+ allosteric effector
●lower pH (more H+)= more stabilized T because higher Kd (more dissociation of oxygen and hemoglobin)
●hemoglobin less likely to bind to Oxygen
●Why? aerobic respiration creates acid (H+) during exercise and we need more O2 to cells. H+ stabilizes T state and oxygen will more efficiently be delivered to cells
Bohr Affect
●Combine H+ and CO2
●CO2 binds (in form HCO3-) to hemoglobin by forming carbamate group at N-term of alpha subunit
●produces another H+ that contributes to reducing hemoglobin’s affinity for oxygen
●Why? hemoglobin carries CO2 to lungs for exhale, won’t bind oxygen, favor T-state
2,3-BPG
●Binds to hole in T state, traps hemoglobin in T state
●One molecule of 2,3-BPG binds to one hemoglobin, prevents oxygen from binding to any of the 4 subunits
● binds to beta subunits with ionic interaction with 3 positively charged residues on each beta subunit (2 subunits)(His2, Lys82, His143)
●Fetal hemoglobin: beta subunit replaced with gamma (y) subunit, His143 mutated to Ser143 -> causes 2,3-BPG to not be able to bind
●WHY? high altitude= want oxygen to be easier to deliver (favor T)
The insulin receptor is an example of what type of protein.
Kinase-Linked Receptor
Which of the following proteins are predominately drug targets for anticancer drugs
Genomic caretaker proteins, kinase-linked receptors, structural proteins
