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BCH210 midterm || with complete solution.
Covalent bond vs. non-covalent bonds in terms of structure correct answers Covalent: hold together amino acids Non-covalent: allow chains to fold into final structure Protein cofactors correct answers non-protein molecules and metal ions that assist in structure and/or function Metal ion cofactors correct answers may interact with the protein or be involved in enzyme catalysis (Ca2+, Co+, Mg2+, Zn2+) Prosthetic groups correct answers typically larger structures; tightly bound molecules by covalent or non-covalent forces Transporters: covalently or non-covalently? correct answers Non-covalently (ex: O2 binding to heme) Cofactors: covalently or non-covalently? correct answers Both Coenzymes correct answers serve as "shuttles" for commonly used functional groups in chemical reactions, so the reaction can occur properly Chainbow image of protein correct answers can visualize in 3D, backwards from blue to red (blue is N-terminus, red is C-terminus) Different types of interactions (5) correct answers 1. Covalent forces
- Ionic/electrostatic/salt bridges
- Hydrogen bonding
- Hydrophobic interactions/effect
- VDW Covalent forces correct answers - strongest
- depends on electron sharing
- nuclei closes together Ionic/electrostatic interactions correct answers - 2nd strongest
- strength depends on polarity of charged species
- aka salt bridge: full charges Hydrogen bonds correct answers - type of electrostatic interaction -- non-covalent
- strength is proportional to polarity of H and other molecule
- N, O, F, Cl, Br
- can occur between molecules or within parts of single molecule Hydrophobic interactions correct answers - depends on entropy of water being released, causing hydrophobic regions to come together
- hydrophobic effect VDW correct answers - weakest
- non-covalent
- depends son size of atoms, distance between them
zwitterion correct answers molecule or ion that has separate positive and negative charges, but an overall net charge of 0 what one letter amino acids don't exist in alphabet? correct answers BJOUXZ amino acids can be metabolized to form what other important molecules? correct answers - hormones
- neurotransmitters
- DNA/RNA
- energy-producing intermediates Disulfides bonds correct answers - covalent interactions
- can be intrachain or interchain
- stabilize structures
- PDI enzymes help catalyze this oxidation reaction secreted vs cytosolic proteins: disulfide bonds correct answers - disulfide bond formation is post- translational modification that occurs in some secreted proteins as they pass through ER
- cytosolic proteins usually contained cystEINES due to reducing nature of cytosol how are disulfide bonds can be broken? correct answers reducing agents in cytosol post-translational modifications (5 groups) correct answers 1. disulfide bond formation (stabilization)
- phosphorylation (stabilization, increase solubility)
- ubiquitination (degradation)
- glycosylation (signaling)
- methyl, acetyl, hydroxyl, carboxyl additions
GFP: aa modification correct answers dehydration and oxidation leads to formation of green fluorophore (after cyclization of ser65-tyr66-gly67)
- protein engineering: change brightness, change color it fluoresces what does aa conservation mean correct answers amino acid segments are properties conserved for structure and/or function pH correct answers -log[H+] at lower pH values meaning? correct answers more H+ present what does pKA measure correct answers strength of an acid low pKa? correct answers low pKa = higher Ka stronger acid choosing buffers around what range correct answers +/- 1 pH unit around pKa choose pKa closest to pH you require at given temperature what is the point of buffers correct answers resist changes in pH so certain functional groups will be maintained (maintain protein structure/function) isoelectronic point (pI) correct answers pH when charge of molecule is 0 how to calculate pI correct answers 1. see where all functional groups protonate/deprotonate
- line up in order
- peptide bonds have partial double-bond character due to resonance, which prevents rotation of peptide bond Phi and Psi angles correct answers rotation is allowed around bonds linking amide and carbonyl to alpha carbon
- phi: amide
- psi: carbonyl What do psi and phi angles minimize? correct answers minimize steric clashing = most stable when functional groups are trans Secondary sequence correct answers Periodic regular structures, folded Alpha helix correct answers - right sided helix with side chains pointing out
- intra-strand HB between i, i + 4
- 3.6 residues per turn what amino acids are typically found in alpha helix? (7) correct answers - glu
- ala
- leu
- met
- gln
- lys
- arg beta strands/sheets correct answers - intermolecular HB (between strands)
- antiparallel, parallel, mixed
- more extended structure = can bring distant parts of protein together
- r chains flip flop sides what amino acids are found in beta strands? (7) correct answers - val
- ile
- try
- cys
- trp
- phe
- thr typically too bulky for alpha helix beta turns correct answers - 4 residue segment that allows peptide chain to turn 180
- found on surface of globular proteins
- can connect secondary structures together (ex: 2 alpha helices, antiparallel beta strands) what amino acids will be found in beta turns? correct answers - pro: common in position 2 bc of turn connecting it back to amine group
- gly (small)
- asn & ser (side chains that can be modified) Tertiary structure correct answers folding of secondary structures into defined protein motifs and domains How do domains function? correct answers can function independently ligands function correct answers can bring distinct regions together
- chemical basis (detergents) considerations of cell lysis correct answers - lysing cells may release proteases, which may cleave protein of interest
- conditions (pH/temp) may alter protein structure
- done in cold rooms to lower activity of possible cleaving
- pH tightly controlled bc many functional groups may get deprotonated/protonated size exclusion/gel chromatography correct answers - proteins separated by size
- column: small molecules enter beads, larger ones elute out
- calibration is required (with known MW) elution volume correct answers fraction where protein comes off column fractionation range correct answers range of MW where you can separate 1 protein from another Vo correct answers - void volume
- anything larger than column's fractional range will go straight through Ve correct answers - elution volume of a molecule (where molecule comes off column) Vt correct answers total volume of column (lowest range of fractionation where smallest molecule spend the most time) area under curve of elution profile correct answers total amount of protein coming off column how to choose column correct answers between vo and vt
protein absorbance correct answers - most molecules are colorless and do not absorb light
- aromatic rings can absorb light and can see how much of aromatic aa is present
- trp absorbs most at 280nm beer lambert law: e correct answers molar extinction coefficient depends on number of aromatic aa present (mainly trp and tyr) ion-exchange chromatography correct answers - separates based on net charge
- cation exchange: elutes catioins common cation exchanger correct answers carboxymethyl common anion exchanger correct answers DEAE how to elute proteins from ion exchange column correct answers - increase salt: compete for binding
- change pH: disrupt HB affinity chromatography correct answers proteins bind based on affinity for certain molecules resin of affinity chromatography correct answers resin contains covalently bound. molecules/ligands that recognizes certain proteins in mixture, interacts via non-covalent interactions how is protein eluted from affinity column correct answers passed through solution of free molecules that compete for binding
WEEK 4 correct answers type 5 membrane protein correct answers identifying: western/immuno blotting correct answers 1. proteins separated by SDS
- primary antibody (specific for protein of interest) added to recognize linear sequence
- secondary antibody (specific for 1st antibody) binds to 1st antibody (produces chemi- luminescent product or fluorescent tag)
- reveals whether or not protein is present, potential size identifying: mass spec correct answers 1. peptides bombarded by high energy electron beam/laser
- attracted to charged plate detector, analyzed for time of flight
- based on charge to mass ratio, can identify protein
- highly charged or smaller molecules will travel faster WEEK 4 correct answers Tryptophan fluorescence correct answers - presence of indole ring allows Trp to fluoresce when excited around 280 nm
- sensitive to polarity Trp red shifted correct answers polar environment - 350 nm trp blue shifted correct answers nonpolar environment - 310 nm purpose of red shift/blue shift correct answers can give an idea of where trp is in protein, cell
when are peaks more intense for trp fluorescence? correct answers when buried in hydrophobic environment chromaphore correct answers chemical groups that absorb UV light (give proteins some color or fluoresce) how do chromaphores allow absorption? correct answers conjugated double bonds -- resonance visualizing 1 and 2 protein structure correct answers IR, CD IR correct answers - vibrating, stretching, bending groups = absorption of IR
- can be used to understand which secondary structures are present which bonds contribute most to IR? correct answers N-H and C==O What lowers the position of IR spectra? correct answers stronger HB = weaker C==O = lower IR position CD correct answers - asymmetry of proteins allow absorption of polarized UV light
- chiral carbons and secondary structures prefer to absorb one direction over other Key wavelengths for CD correct answers - alpha helices: 208 & 222 nm (neg), 195 (pos)
- beta sheet: 217 nm (neg)
- random coil: 198 nm (neg) how to see CD properly? correct answers needs to be in hydrophobic/sds SOLUTION for protein to properly fold
- diffraction can be used to elucidate structure single particle cryo-Em, what can you do with multiple structures? correct answers - single particle structure can be obtained
- multiple structures can be averaged out to build 3D representation benefits of cryo vs nmr correct answers cryo can visualize larger protein structures studying protein-protein interactions: pull down assays correct answers - does x interact with y?
- pull down x to see if y comes with it
- used with western blotting western blotting & pull down assays correct answers - pull down x with specific antibody 1
- use western blotting and use specific antibody 2 to see if y is present
- can also use sds page protein-protein interactions: chemical cross-linking correct answers - form covalent bonds in side groups between molecules
- reveal inter and intra organization what reactive side chains can be targeted with cross-linking? (5) correct answers 1. primary amines
- carboxyls
- carbonyls
- sulfhydryls
- form disulfide bonds
how to adjust crosslinker? (6) correct answers 1. length
- cleavability
- how to purify
- composition of two dimers
- reactive groups
- bifunctional (homo vs hetero) crosslinker: biotin correct answers can detect or purify any cross-linked molecules how to detect cross-linkers? correct answers - western blot, mass spec, sds cross-linkers and x ray crys correct answers cross-linkers can help stabilize structure for x ray crys protein-protein interactions: 2 hybrid systems correct answers - fusion proteins can be made to include a pair of known protein interactors
- bait protein interacts with captured prey -- reporter can be used to detect interaction 2 hybrid system example correct answers MYTH (yeast membrane proteins) designing fusion proteins correct answers - tags of stretches of aa can be introduced, or tags/larger proteins
- affinity chrom can be used to isolate fusion proteins 2 hybrid example: BioID correct answers - uses birA to biotinylate nearby proteins
- can be purified using streptavidin beads, identified using wb or mass spec
- lower km = tighter binding where do cells normally keep concentrations near? correct answers near km or kd so can adjust to fluctuations limitation of MM equation correct answers only estimation of vmax and km Lineweaver-Burk Plot equation correct answers Turnover number correct answers kcat, used to describe enzyme efficiency (constant when 100% of enzyme is completely saturated) larger kcat =? correct answers faster production of product kcat equation correct answers kcat = vmax / [E]total enzyme: oxidoreductasees correct answers redox reactions (ex: alcohol dehydrogenase) enzyme: transferases correct answers group transfer between molecules (ex: protein kinase a) enzyme: hydrolases correct answers hydrolysis reaction (sucrase) enzyme: lyase correct answers addition/cleavage reactions (adenylyl cyclase) enzyme: isomerases correct answers group transfer within molecule (creates isomer, phosphoglucomutase)
enzyme: ligase correct answers join 2 molecules using nucleotides (atp, gtp) enzyme: translocase correct answers movement of ions or molecules across. membrane are all enzymes very specific? correct answers no, some can be non-specific What does Km indicate at 50% binding? correct answers concentration of substrate when half active sites are bound WEEK 5 correct answers essential membrane functions (3) correct answers - compartmentalization to increase cellular efficiency
- barrier regulating important and export of molecules
- signaling Fluid mosaic model correct answers - fluid: membrane components. can move quite rapidly in the plane of membrane
- mosaic: diverse mixture of molecules in membrane recent modifications of fluid mosaic model correct answers not as fluid as originally thought to due to lipid rafts, cytoskeleton, and other TM proteins how is membrane asymmetric? correct answers - different composition of different lipids on each bilayer Where are glycolipids and glycoproteins found? correct answers extracellular surface -- stay outside bc movement through hydrophobic tails is energetically unfavorable
