Exam 1

peptide bond

-formation - condensation of 2 AAs to form a dipeptide -peptide bond has no charge

determination of protein folding patterns

-AA sequence -phi/psi bonds -hydrophobicity/hydrophobic effect - hydrophobic R groups will wanna be inside/away from water, hydrophilic outside -> driven by entropy

chemical properties of water that contribute to its solvent properties

-1. polarity and H bonding -2. reversible ionization

coordination sites for Fe2+

-2 coordination sites -5th site - proximal histidine -6th side - binds O2 -proximal histidine comes from one of the alpha helices that is Mb/Hb structure -distal histidine comes from another alpha helix -oxygen bonds to hydrogen of distal histidine (H bond) -> stabilizes distal histidine -CO bonds to iron irreversibly (why it kills us) but distal histidine interferes w binding of CO for most part -steric hindrance bc CO is so big -> distal histidine doesn't rly fit w CO

proteins w quaternary structure

-2 or more polypeptide chains stabilized by noncovalent interactions -some proteins have subunits linked by disulfide bonds -most subunits interact by noncovalent interactions to form the functional protein

restricted rotation around peptide bond

-3 main chain rotational degrees of freedom for each dipeptide unit in a protein - phi, psi, and omega -rotation around peptide bond omega is restricted bc peptide bond has partial double bond character -omega is partial double bond between C and N

important characteristics of RNase

-4 cysteine bonds - harder to break than other bonds (stable) -small, heat stable protein -enzymatic activity -single polypeptide chain (tertiary structure) w disulfide bonds

factors that determine how a protein will fold

-AA sequence -hydrophobic effect -specificity

tyrosine

-amphipathic -both polar and nonpolar aspects -ring is hydrophobic -OH can H bond

directionality of peptide chain

-AA sequence (primary structure) is always written from amino terminal to carboxyl terminal (left to right) -polypeptide chain consists of repeating part called main chain/backbone and a variable part consisting of distinctive AA side chains -backbone has H-bonding potential bc of carbonyl groups and H atoms that are bonded to N of amino group

optical activity of AAs

-AAs are optically active -alpha-C is chiral center of molecule -exception is glycine (R = H) -only the L configuration is found in proteins

types of noncovalent interactions

-H bonds -electrostatic, ionic interactions -van der Waals forces -hydrophobic interactions -^these can dictate structures and then functions of molecules in cells

beta sheets

-H bonds link 2 or more beta strands in a beta sheet -differences in directionality and H bonding geometry -H bonds are perpendicular to strands -extended structure, large phi and psi angles -R groups extend above and below plane -> accommodates bulky R groups

effect of H+ and CO2 on O2 binding to Hb

-Hb carries 2 end products of respiration (CO2 and H+) -rxn is spontaneous but dissolved CO2 reacts slowly w H2O to form bicarb and H+ -rate is increased by carbonic anhydrase -making pH more acidic shifts curve to right -> less binding affinity for O2 -more CO2 also shifts curve to right -> less binding affinity for O2

Keq of water

-Keq of water = 1.8x10^-18 -reactant (H2O) is v much favored -actual # of dissociated molecules is v small relative to # of undissociated -conc of H2O is considered unchanged

ion product of water (Kw)

-Kw = 1x10^-14 = [H+][OH-] -if [H+] increases, then [OH-] decreases -pure water is electrically neutral so it can be rewritten as Kw = [H+]^2 = 1x10^-7 -this property of water is central to role as biological solvent

O2 binding curves for Hb and Mb

-Mb is hyperbolic (monomer) -Hb is sigmoidal (S shaped; tetramer -> can have allosteric cooperation and binding changes as a result) -once one O2 is attached to Hb, it's easier for more to bind bc conformation of 4 subunits changes -at p50 (pO2 corresponding to half saturation), Mb has much higher affinity for O2 (binds more tightly than Hb) -p50 indicates affinity for O2 -Hb binds O2 v well at high pO2 (100 torr) found in lungs -in tissues, pressure drops down around 25 torr and Hb drops affinity for O2 but Mb still grabs onto it -in tissue, Hb gives up O2 to Mb so that O2 ends up in muscles for energy production

reason why heme is used for O2 binding

-O2 is poorly soluble in water (diffusion thru tissue is ineffective) -transition metals have strong tendency to bind O2 (v reactive in free form, esp iron) -iron must be sequestered to render it less effective (heme group in Mb and Hb structures)

common electronegative atoms

-O>N>S -give rise to polar covalent bonds (electrons not equally shared between atoms) -relatively reactive bc of polarity

acids and bases

-acid is proton donor -base is proton acceptor -acids ionize to form proton and base -chemical formed upon ionization of acid is called conj base -acid formed when base binds proton is called conj acid -extent of ionization determines acid strength (Ka = acid dissociation constant)

2,3-bisphosphoglycerate (2,3-BPG)

-affects oxygen binding to Hb -AKA 2,3-DPG -v negatively charged, polar -binds in central cavity bc it's negatively charged -> there are lysine, histidines in central cavity which are (+) charged -2,3-BPG stabilizes T state -when O2 binds subunits conformational change occurs and central cavity is no longer open -> 2,3-BPG doesn't fit anymore so it gets kicked out -noncovalent interactions stabilize 2,3-BPG in central cavity -shifts Hb curve to right, decreases O2 affinity

causes of protein misfolding

-age can increase changes -mutation -high/low pH or temp can make it more favorable for AAs to denature/refold poorly

nonpolar R groups (hydrophobic)

-aliphatic - branched chain -aromatic - rings

ionization of AAs

-all AAs have at least 2 pKa values -alpha-carboxyl group has pKa of 2-3 -alpha-amino group has pKa of 9-10 -group becomes deprotonated when pH = pKa -zwitterion (carboxyl is deprotonated, amino is protonated) is at normal pH

general structure of an AA at pH = 7

-all AAs have carboxyl (-COOH) and amino group (-NH2) -at pH = 7, carboxyl group is deprotonated and amino group is protonated -if R chain isn't charged, these 2 charges cancel out -there are 20 common AAs -side chain is what differentiates AAs

dihedral/torsional angle

-angle between 3 atoms in a plane and the 4th atom coming out of the plane -diff torsional angles lead to diff conformational energies -as you rotate around dihedral angle, bonds become higher energy and less stable, and vice versa -basically a new Newman Projection

polar charged negative AAs

-aspartate -glutamate -these are acidic AAs -can contribute to buffering capacity inside cell

chemistry of CO2 binding

-at low pH and high CO2 in peripheral tissues, Hb affinity for O2 decreases -CO2 binds to alpha-amino group at amino terminus of each global chain -amino terminus becomes an anion -reacts w Arg141 of alpha chain; forms a salt bridge -deoxygenated (T) state stabilized by this and promotes release of O2

general pH and pKa rule

-at pH<pKa, H+ is on, AA is protonated -at pH>pKa, H+ is off, AA is deprotonated

relationship between O2 dissociation curve and delivery of O2 in the body

-at pO2 = 100 in lungs, Hb and Mb bind O2 similarly -at pO2 = 20 in tissues, Hb is 66% likely to give up O2, Mb is only 7% likely to give up O2

ways to simplify protein structure

-atoms represented as spheres -bonds represented as sticks -main chain (backbone) only, no side-chain R groups -trace (centers of AAs are connected by "virtual bonds") -ribbon cartoon (virtual bond lines between AAs are smoothed; most common diagram) -in ribbon, alpha helices are red, beta sheets are yellow arrows (direction of arrows tell you which way AA sequence runs)

electrostatic and ionic interactions

-attraction/repulsion of charges -occur between atoms w a complete negative and complete positive charge (water is just a partial charge) -e.g. NaCl -also called salt bridges -ionic interaction can be disrupted by water (soluble in water) -strength of interaction determined by distance and solvent (e.g. ionic interactions in NaCl would be stronger in butanol than water bc water is more polar and NaCl wouldn't really dissolve in butanol) -important for protein-protein interactions, DNA-protein interactions, catalytic mechanisms

classification of AAs

-based on chemical nature of R groups (they can engage in noncovalent interactions that determine protein structure/function) -R group confers ability of AAs to vary in charge, shape, size; ability to H bond, hydrophobicity; chemical reactivity -e.g. Trp has hydrophobic R chain, can't H bond; Glu has charge, can H bond

basis for organization and complexity

-basic characteristic of life is a high degree of order (e.g. cells have membranes, nuclei inside, DNA inside nuclei) -basis for organization is water

van der Waals forces

-basis - transient asymmetry in electron distribution of 1 molecule will induce complementary asymmetry in a nearby molecule (i.e. induced dipole-induced dipole) -result is charge fluctuations -> transiently produced (+) or (-) poles even in nonpolar molecule (TEMPORARY) -distance matters -> present w "snug fit" between atoms -3-4 angstroms is ideal distance for best attraction -too far/too close can make them repulsive -weakest of noncovalent interactions (.1-1 kcal/mol)

shift with O2 binding

-binding of O2 induces a 15˚ shift along interface -goes from deoxyhemoglobin to oxyhemoglobin -size of central cavity changes on binding oxygen -T state to R state -Hb is allosteric - has 2 diff stable shapes depending on binding of O2 -when O2 binds, there is a slight shifting that occurs of where everything is in conformation w each other; "pocket' in the middle of the molecule disappears when shift happens

pH regulation

-body compensates for generation of acid due to metabolic activity -blood pH must be maintained in narrow range (7.36-7.44) -intracellular pH = 7.1 (range of 6.9-7.4) -pH homeostasis is v important to maintain normal structure and function of molecules

ketoacidosis

-body makes a lot of ketones as it consumes fats to get energy -> unregulated buildup of ketone bodies acidifies body

lactic acidosis

-buildup of lactic acid after exercising

pHLIPs as diagnostic/therapeutic delivery agents

-cancer cell EC environment causes pHLIPs to become embedded in membrane -can mark cancer cells on N terminus to tell difference from healthy cells w label/antigen -> helps w targeting -can conjugate toxin/drug to C terminus -> can get cleaved off of pHLIP and kill cell/disrupt cell activity

buffering capacity of Hb

-carbonic acid (H2CO3) is major metabolic acid produced in body and is a major buffer -ability to serve as a buffer due to dissolved CO2 in body fluids which is 500x more than H2CO3 -dissolved CO2 is in equilibrium w air in lungs; availability increases/decreases by rate of breathing -get rid of CO2 by breathing it out -Hb can bind CO2 and H+ and take them to lungs to get rid of them, or CO2 travels to lungs in form of bicarb -increasing respiration increases O2 in tissue -> increases metabolism -> increases CO2 and protons -Hb binding CO2 and protons contributes to buffering capacity of blood

thalassemias

-caused by loss/substantial reduction of single Hb chain -alpha-thalassemia - alpha chain isn't produced in sufficient quantity -> tetramers of beta chain form (HbH) and bind O2 w high affinity but no cooperativity -beta-thalassemia - beta chain isn't produced in sufficient quantity -> alpha chains aggregate and precipitate, leading to loss of RBCs and anemia

gastroesophageal reflux disease (GERD)

-chronic digestive disease -develops when stomach acid refluxes into esophagus -backwash of acid (often feels like heartburn) irritates lining of esophagus by exposing tissue to v acidic conditions (pH of 1-2) -risk factors - smoking, obesity, etc

contours in a Ramachandran Plot

-combines 2 dihedral angles of freedom into 3D map -the deeper the map, the more favorable the conformation -experimental Ramachandran plot used distribution from 470 actual protein X ray structures, matched his prediction -predicts where alpha helix/beta strands will be favorable

RNase experiment

-control - measure RNase activity -1. treat RNase w urea and excess beta-mercaptoethanol (beta-m breaks apart cysteine disulfide bonds over and over again) -2. 8M urea is a denaturing agent -> unfolds protein (denatures all noncovalent interactions between proteins) -3. beta-mercaptoethanol (strong reducing agent) disrupts disulfide bonds -4. these reagents together completely unfold structure; peptide bonds remain intact -5. remove urea and beta-mercaptoethanol by dialysis (taking away beta-mercaptoethanol leaves you w protein w random disulfide bonds formed [1% chance of correct disulfide bond pairings]) -no activity after treatment w urea and beta-mercaptoethanol bc it loses its structure, so it loses its function -adding trace beta-mercaptoethanol leads to native RNase structure again -after removal of both agents, RNase becomes scrambled (1% chance that cysteine bonds will bond correctly) -> you add trace beta-mercaptoethanol which will unbound and bound cysteine bonds over and over again til it can go back to native state and then it'll stay there bc that's the lowest energy conformation -if you had only added urea, protein would be denatured but disulfide bonds wouldn't break

covalent vs noncovalent bonds

-covalent - strong, hold together molecules, stable -noncovalent - weak, interactions w/in and between molecules, specific -both stabilize folded proteins -advantage of noncovalent bonds is that they're weak bc they can break and form easily

determination of polarity

-depends on arrangement of electrons in covalent bond

pH scale

-derived from ionization of water and is logarithmic -pH = -log[H+] -difference of 1 pH unit means that solution has 10x difference in [H+] compared to the other bc it's a log function -pH ranges from 0 (v acidic) to 14 (v basic)

loss of bicarb

-diarrhea -altered kidney function -can be due to decrease in production of acid

forces contributing to stability of native state

-different physical forces, which sometimes counteract and compensate one another, contribute to stability of native state of proteins -conformational (chain) entropy -van der Waals interactions -electrostatic interactions between charged and polar groups -hydrophobic effect

reversible ionization

-dissociation of water -results from nucleophilic attack by O on proton of adjacent water molecule -water prefers to be in full molecule, not dissociated

hydrophobic effect and protein folding

-dissolution of polar solute in water is favorable -dissolution of nonpolar solute in water is unfavorable -water has to form an ice-like clathrate structure around nonpolar solutes (this is entropically unfavored) -nonpolar solutes prefer to interact w each other than w water bc this reduces contact surface of solute w solvent -i.e. hydrophobic side chains like Val, Ile, Leu, Phe would tend to be buried in protein interior -water forces protein folding so it can minimize # of clathrate structures it has to form around exposed hydrophobic groups in an unfolded protein -> this gets around the idea that denatured proteins are more entropically favored

doxorubicin

-drug to treat cancer -pKa = 8.2 -has ionizable primary amine -v hydrophobic molecule so it can diffuse thru cell membrane pretty passively whereas ionized molecules have to enter more actively -when doxorubicin is ionized, cell cannot take up as much of it -using Henderson-Hasselbalch, more of the doxorubicin enters normal cells (17%) vs cancer cells (5%)

titration curve

-each pt on curve represents diff ratio of dissociated to undissociated weak acid -as curve gets more basic, there is more conj base -when curve is more acidic, there is more acid -when [HA] = [A-], acid is 50% dissociated and pH = pKa -midpoint is best buffer pt (where [acid]=[conj base], pH = pKa) -start w fairly acidic -> add hydroxyl equivalent -> pH goes from acidic to basic -slope is flatter in middle and pH change isn't much -> rises fast at end bc you're losing buffering capacity

ionizable side chains

-enhance reactivity and bonding -7/20 AAs have ionizable side chains depending on side chain pKa and pH -tyrosine and cysteine -arginine, lysine, and histidine are typically basic -aspartic acid and glutamic acid are typically acidic -these AAs are able to form ionic bonds and donate/accept protons to facilitate rxns

nonpolar

-equal sharing -uniform charge distribution -e.g. methane

importance of polarity

-explains why molecules are hydrophilic/hydrophobic -importance of water as an organizing principle in bio -leads to noncovalent interactions (e.g. interactions that have to do w polarity between AAs)

Ka

-expresses extent of ionization -acid dissociation constant/equilibrium constant for dissociation -constant for any given acid -if Ka is large, a strong acid is indicated -some acids have 2-3 diff Ka values bc they have more than 1 ionizable group (some Hs come off more easily than others) -tells you when ionizable side group is coming off

selective advantage of outer acidic environment for cancer cells

-favors proliferation -avoids apoptosis -favors migration and invasion (the more aggressive/invasive a tumor is, the more acidic it is) -avoids immune detection

fetal vs adult hemoglobin

-fetal has 2 alpha chains and 2 gamma chains -adults have 2 alpha chains, 2 beta chains -serine substitutes for His143 in fetal Hb -serine is polar uncharged, His is (+) charged -> 2,3-BPG is not quite as attracted to central cavity so it comes out more easily -> T state isn't as stable so you can go to R state more easily -> fetal Hb binds O2 more tightly than adult Hb -ultimately you wanna give fetus more O2 -O2 flows from maternal oxyhemoglobin to fetal deoxyhemoglobin

protein stability

-folding produces 2 opposite forces - decrease in conformational entropy, minimization of protein's solvation energy -deltaG for typical protein is 5-10 kcal/mol -marginal stability of proteins -> turnover when no longer needed

important features of peptide bond

-formed by condensation rxn involving elimination of water -equilibrium favors reactants vs products - energy consuming process -partial double bond character - resonance -covalent; kinetically stable -planar in geometry and rigid -trans configuration

sickle cell disease

-genetic mutation resulting in substitution of valine for glutamate at position 6 of beta chains -can be fatal when both alleles of beta chain are mutated -in sickle-cell trait, one allele is mutated and one is normal (these individuals are asymptomatic)

nonpolar aliphatic AAs

-glycine -alanine -valine (branched chain) -leucine (branched chain) -isoleucine (branched chain) -methionine (thioether) -proline (ring structure but not aromatic)

ramachandran plot

-graphs psi as a function of phi -colored part of graph indicates favored products -box w nothing indicates lots of steric hindrance -> you never get this structure -2 favored conformations contribute to secondary structure of AAs

covalent bonds

-happen everywhere -high energy bonds -electron sharing

background on Anfinsen's ribonuclease experiment

-he asked "what determines how a protein will fold?" -start w protein that has 4 disulfide bonds -> add a catalyst -cysteine is unique bc can form disulfide bonds across chain -protein has 8 cysteines, all paired in disulfide bonds

important structural features of heme

-heme = protoporphyrin ring and Fe2+ -protoporphyrin ring is hydrophobic, planar -4 Ns stabilize the Fe2+ and prevents Fe2+ to Fe3+ thru their electron donating capacity (Fe2+ binds O2 reversibly, Fe3+ cannot) -heme is located w/in hydrophobic pocket of structure -> prevents full transfer of electrons to give irreversible oxidation (key to binding/release of O2) -free heme doesn't bind O2 reversibly

important characteristics of the peptide bond

-hybrid of 2 resonance states -C-N bond has partial double bond character -free rotation doesn't occur -rigid -planar geometry (6 atoms [alpha-C, C, O, N, H, and alpha-C] lie in a plane) -exist in trans configuration to avoid steric hindrance between R groups

phospholipid bilayer

-hydrophilic and hydrophobic properties -phospholipids are called amphipathic/amphiphilic molecules; form membranes when exposed to water -phospholipid has polar head group and hydrophobic tails -> in water, bilayers form spontaneously so heads are exposed to water and tails are protected on inside -throw phospholipids into water and shake -> tiny bubbles of lipid bilayers form w polar head group on inside and outside where water is (lipid micelles) -affects which proteins are inserted into membrane (e.g. structure, polarity, etc)

water and the hydrophobic effect

-hydrophobic effect is clustering of hydrophobic molecules -entropy-driven association -hydrophobic interactions form spontaneously

Hb binding and release of O2

-hydrophobic pocket in Hb and Mb structure is key to understanding binding and release of O2 -structure prevents full transfer for electron to give irreversible oxidation of iron thus promoting reversible O2 binding -iron is only partially oxidized in heme -if iron is not stabilized, O2 can steal electron and make it so that iron is fully oxidized (+3 form) -> then it cannot bind more O2 and O2 becomes superoxide

pKa of side chains

-if R group ionizes, that AA will have a 3rd pKa -7/20 AAs (tyrosine, cysteine, arginine, lysine, histidine, aspartic acid, and glutamic acid) have readily ionizable side chains -if pH<pKa, then ionizable group will be in acidic form -if pH>pKa, then ionizable group will be in basic form

peptide torsional degrees of freedom

-if omega angle across peptide bond is fixed, each AA has only 2 backbone torsional degrees of freedom - phi and psi -phi is between alpha C and N -psi is between alpha C and C

hemoglobin and myoglobin

-illustrate important concepts like relationship between protein structure and function, importance of noncovalent interactions (reversible binding of a ligand to a protein), and buffering capacity -these 2 proteins are evolutionarily related, share sequence homology -first proteins crystallized to determine structure by X ray crystallography -both contain heme prosthetic group (prosthetic groups are permanently associated w protein and contribute to its function)

side chains/R groups

-important functional groups which take part in noncovalent interactions -if functional group accepts/donates a proton, it can engage in acid-base catalysis during rxns -pKas are also important -polar charged R groups can contribute to buffering capacity inside cell

carbonic acid/bicarb buffer

-important in blood -carbonic acid/bicarb system works bc of high [CO2] dissolved in body fluids and equilibrium between dissolved CO2 and CO2 in lungs -pKa = 6.1

hydrophobic effect

-inability of water to dissolve nonpolar molecules results in this important organizing principle -hydrophobic molecules tend to cluster together in aqueous solutions -> they don't dissolve in water bc they're nonpolar -powered by increase in entropy of water that results when hydrophobic molecules come together -powerful organizing force in biological systems -major determinant of how a protein folds

phosphate buffer

-intracellular fluids -pKa = 7.2

amyloid

-occurs when misfolded protein can't be removed from cell -highly ordered -beta strands to fiber axis (transition from normal alpha-helix to abnormal beta-sheet conformation) -extensive H bonding -> v stable bc lots of H bonds between sheets -importance of side chain interactions in aggregate formation -self assemble into fibers -resistant to degradation -steric zipper - complementary pairs of beta-sheet structures formed by short peptide chains

hydrophobic interactions

-involve nonpolar molecules which can't form H bonds w water -water can form H bonds w adjacent water molecules -water can only interact 3-dimensionally around nonpolar molecules bc it has to be ordered -> when you introduce more hydrophobic molecules, the hydrophobic molecules will come together which frees up some water molecules bc less hydrophobic molecules to interact w -> water becomes more entropic and can make their transient H bonds, be chaotic, have more energy -energetically favored for nonpolar molecules to come together

secondary structure

-involves (1) H bonds between NH and CO along polypeptide backbone and (2) preferred Ramachandran angles -alpha helix, beta sheet, turns, irregular structure -^each one of these 4 accounts for ~25% of total protein structures (i.e. turns and irregular structures are as common as alpha helices and beta sheets)

polar charged positive AAs

-lysine -arginine -histidine -these are basic AAs -can contribute to buffering capacity inside cell

entropy

-measure of disorder -favors the denatured state -native state (N)/compact structure has small entropy due to well defined unique conformation -denatured state (D)/noncompact structure has large entropy due to large # of diff conformations

entropy (S)

-measure of disorder -possible arrangement of molecules -makes hydrophobic interactions favorable -ordered state is low entropy -disordered state is high entropy -entropy drives interaction between nonpolar molecules bc hydrophobic interactions allow for increase in entropy of water

proteasomes

-mechanism to eliminate protein misfolding -degrade proteins to free AAs -> AAs are recycled to be used again

chaperonins

-mechanism to eliminate protein misfolding -needed to accelerate slow steps using ATP (since info for folding is inherent in sequence already) -unravel misfolded proteins -prevent aggregate formation -first identified as heat-shock proteins (hsp60, hsp70, hsp90, etc) -separate proteins from elements that would disrupt foldings

chemical/physical factors that drive folding

-minimum criteria applies to all proteins: -AA sequence (primary structure; phi and psi angles minimize steric effects; correct pairing of cysteines for disulfide bonds) -hydrophobic effect (bury the hydrophobic side chains, minimizing contact w water; most polar residues face the outside of the protein and interact w the solvent) -retention of partially folded correct intermediates (related to free energy; lowest energy state is native state)

buffers

-mixture of undissociated acid and its conj base -e.g. acetic acid and acetate ion -consequence - causes solution to resist changes in pH when either H+ or OH- is added -> structures and functions of macromolecules, proteins, etc will stay stable -proteins (AAs) are also buffers

energy landscapes

-multiple microscopic folding pathways -random search funnel -downhill funnel - native-like interactions are kept and promoted -rough funnel - allows for trapped non-native conformations (ridges)

function of hemoglobin

-must bind O2 in the lungs, release it in the capillaries -when O2 binds to Fe in heme of one Hb subunit, Fe is drawn into plane of porphyrin ring -> disrupts key noncovalent interactions in that subunit which causes a change in conformation -O2 moves up into plane of hemoglobin itself

disruption of noncovalent interactions on binding O2

-no oxygen = T state -oxygen = R state -as O2 binds to Fe, F helix moves -there is a loss of interactions between F and H helices: -1. salt bridge between Asp94 and His146 is broken -2. salt bridge between Lys40 and His146 is disrupted -3. H bond between Val98 and Tyr145 is disrupted -small conformational changes happen but entire molecule doesn't fall apart bc there are other noncovalent interactions occurring

reverse turns and loops

-no repetitive H bonding like in helix and sheet -lie on protein's surface, participates in interaction -enables change of direction in protein/connects strands

importance of R groups

-noncovalent interactions contribute to protein folding and reactivity -effect of mutations which result in AA substitutions -conserved vs non-conserved substitution -noncovalent interactions between R groups in proteins determine structure

reverse pH gradient of cancer cells

-normal differentiated cells have IC pH = 7.2, EC pH = 7.4 -cancer cells have IC pH ≥ 7.4, EC pH = 6.7-7.1 -this is bc cancer cells under go anaerobic respiration - break down glucose and produce lots of lactic acid -cancer cells don't wanna be acidic on inside of cell bc they still need to do their cellular activities and proteins need to work -> they pump protons outside cell and create acidic environment

metabolism and changes in [H+]

-normal, daily metabolism produces changes in [H+] -metabolism results in 13-22 moles of acid produced/day (if they were strong acids which dissolved in water, pH would be <1) -most acids produced are weak organic acids (HA) which undergo partial ionization in water (solvent)

mutation

-not responsible for misfolding in all cases -sickle cell anemia is caused by single AA substitution in beta-chains of Hb (valine replaces glutamic acid) -> results in aggregation of Hb molecules and formation of insoluble fibers that result in sickle shape -glutamic acid is (-) charged polar, valine is nonpolar (hydrophobic replaces hydrophilic) -> forms insoluble fibers which ruin shape of RBC -sickle cell evolved to help boost malaria immunity

H bonds

-occur between EN atom (acceptor; e.g. O) and H atom that is covalently bonded to another atom (donor) -weak association (1-5 kcal/mol, covalent bond is 50-200) -rapidly form, break, reform in diff orientations -important for solvent properties of water, structure of proteins and nucleic acids (e.g. DNA and RNA) -A and T, G and C form H bonds between each other to make double helix -> this can break so DNA can open up -water forms H bonds w other water molecules (avg H bond lasts 10 picosecs) -difference between diff states of water is due to H bonds (e.g. boiling breaks H bonds)

Henderson-Hasselbalch equation

-pH = pKa + log [A-]/[HA] -only applies to weak acids and bases (not strong acids bc they completely dissociate in water) -large Ka or small pKa indicates strong acid -pKa = -log Ka -Ka is a constant so it doesn't change -pH changes depending on dissociation of acid -equation describes extent of dissociation of weak acid at any pH and can be plotted as titration curve

small changes in pH as signaling mechanism

-pH increases of .2-.3 units promote cell proliferation, migration, assembly of actin filaments -physiological changes in pH regulate specific proteins (pH sensors) -change in binding affinity, activity, recruitment to membranes -e.g. ion channels, pumps, enzymes -subtle changes aren't catastrophic, they can help body function properly -increase of intracellular pH can be associated w cancer, decrease can be associated w several neurodegenerative diseases -molecules in body depend on neutral pH to keep normal structure and function

metabolic acidosis

-pathological acid/base disorder -increase in production of acid or loss of bicarb -can be a complication of drugs or other exposures

formation of a peptide bond

-peptide bond is covalent -occurs between alpha-carboxyl and alpha-amino in 2 AAs -all AAs can take part in peptide bond -type of rxn is condensation -water is eliminated -rxn is not favorable (requires input of energy by cell) -rxn is carried out by catalytic RNA located in ribosome

rotation about phi and psi angles

-permits protein to fold in different ways -not all combos are permissible due to steric hindrance -restricted set of phi and psi angles limits # of possible protein structures -steric exclusion is an organizing principle for protein structure

nonpolar aromatic AAs

-phenylalanine -tryptophan -tyrosine (amphipathic) -aromatic ring structure permits absorption of UV light (280 nm)

phi and psi bond angles

-phi - angle of rotation about bond between N and alpha-C -psi - angle of rotation about bond between carbonyl and alpha-C -rotations occur w/in one AA

solvent properties of water

-polarity, EN decide what's soluble in water -e.g. lysine, D-glucose are soluble in water -fatty acids are insoluble

levels of protein structure

-primary - AA sequence -secondary - regular backbone H bonding patterns (e.g. alpha helix, beta sheet) -tertiary - complete and independent protein structure (whole polypeptide) -quaternary - aggregate/complex of 2 or more proteins -possible to have secondary w/o tertiary, descriptions are for organizational purposes -the more protection of Hs, the more stable the H bonds -Kaj Linderstrom-Lang did lots of work on this in Carlsberg labs, Copenhagen

primary structure

-protein is a chain of AA monomers -proteins = uniform backbone, genetically encoded side chains (R groups) -mainchain/backbone is same for every polypeptide; is polar (N terminus is positively charged, C terminus is negatively charged) -difference between polypeptides is R groups on AAs

protein misfolding and disease

-protein misfolding which exceeds cell capacity for removal results in diseases -e.g. neurodegenerative diseases like Alzheimer's Parkinson's, Huntington's; type II diabetes -alpha-beta-fibrils from Alzheimer's diseased brain look different than normal -prion protein - membrane protein found in brain -misfolding prion proteins associated w mad cow disease, Creutzfeldt-Jakob disease (inherited point mutation), and sporadic forms

thermodynamic hypothesis

-proteins will fold on their own into their native state (least energy state) -the 3D structure of a protein in its normal physiological milieu is the one in which the Gibbs free energy of the whole system is lowest, i.e. the native conformation is determined by the totality of interatomic interactions and hence by AA sequence in a given environment -corollary - if we understand what determines free energy minimum, we should be able to determine structure from AA sequence

chemistry of H+ binding

-protons react w His side chains -His146 in beta subunit is important -> protonation promotes release of O2 -pKa of histidine can be affected by neighbors (pKa of His 146 = 6.6 for oxygenated Hb, 8.2 for deoxygenated Hb) -proton can come in from the tissues and reform salt bridge between beta-His146 and beta-Asp94 -> this stabilizes T state -reformation of salt bridges upon Hb release of O2 (same salt bridges that were disrupted on binding O2)

allosteric behavior of Hb

-quaternary structure -different conformational states (R and T) -cooperativity leads to change in conformation -change in conformation of one heme subunit that binds O2 pushes around conformation of other heme subunits -> move into shape that makes them more able to pick up O2 -sigmoidal binding curve/kinetics

equilibrium constant (Keq)

-ratio of [product] to [reactant] -for water, Keq = [H+][OH-]/[H2O] -if [product]=[reactant], then Keq = 1 and rxn is at equilibrium -rate constants for forward and reverse rxns are equal -concs of reactants and products have no net change over time -if Keq>1, then [product]>[reactant] (means forward rxn is favored)

Bohr Effect

-regulation of O2 release from Hb by H+ and CO2 -Hb can bind H+ and CO2 -Hb is considered a pH sensor

misfolding

-retains structure -incorrect structure -can result in loss of/acquired (new) activity -a misfolded protein can refold, degrade to AAs, or form aggregates/amyloid fibers -> leads to disease -causes include mutation and exposure of amyloid segments

polar uncharged AAs

-serine (OH group; almost always protonated) -threonine (OH group; almost always protonated) -tyrosine (OH group) -cysteine (SH group; v reactive) -asparagine (carboxamide; can H bond) -glutamine (carboxamide; can H bond)

pH low insertion peptides (pHLIPs) and cancerous tissue targeting

-short peptides (~30 AAs long) -protonated AAs will affect structure -lots of protonation in highly acidic EC environment -> becomes protonated (now neutral) and therefore more hydrophobic -> enters membrane due to increased affinity for hydrophobic core -> takes different, helical shape bc of diff AA interaction and forms transmembrane helix -> once it gets into cell where pH is more neutral, it's deprotonated (now negative) and anchors the pHLIP in the membrane -can conjugate a drug/toxin to C terminus (ends up inside cell); a dye/antigen to N terminus (ends up outside cell)

exposure of amyloid segments

-short sequences of ~6 AAs -present in large % of proteins -normally buried w/in interior of structure, but can become exposed -results in aggregation and formation of insoluble fibers

titration curve and pKa of histidine R group

-side chain of histidine is an imidazole group -can bind/release protons at neutral pH, depending on environment (i.e. can act as acid/base) -histidine is found at active sites of many enzymes that require a proton donor/acceptor -ionization is v close to physiological pHs -> cells can take advantage of this -v similar to EC cancer cell pHs -at pH = 7.3, 95% is [His], 5% is [His+]

significance of sigmoid curve shape

-sigmoid shape of O2 saturation curve indicates cooperativity between subunits in binding O2 -binding of O2 to one subunit disrupts some noncovalent interactions while forming new ones -alteration in conformation makes it easier for oxygen to bind to other subunits

function of buffers

-simple view - combines H+ and OH- and converts them to a nonionized form -complex view - involves 2 reversible equilibria, one involving water

what makes water essential for life

-solvent for rxns and interactions (typical eukaryotic cell is 65-70% water, human body is 65% water) -reactant/product in rxns (e.g. use water as reactant to put together molecules, break apart glucose to get energy and water as products) -organizing principle

alpha helix

-stabilized by H bonds -each peptide bond is connected by H bonds to a peptide bond 4 AAs away -periodicity of 3.6 residues (i.e. 3.6 AAs per turn) -H bonding - each carbonyl O is H bonded w amide (N-H) -compact structure, small phi and psi angles -phi and psi are similar (phi = -57˚, psi = -47˚) -R groups project outward

myoglobin

-stores O2 in muscle -single polypeptide chain; monomer -153 AA -17,200 MW

relationship between protein structure and function

-structure determines function -functional versatility requires structural versatility -175,000 protein structures in PDB as of 9/12/21 -can be categorized in ~1000 diff folds/shapes -goes from linear sequence to folded structure

amyloid formation

-thought to be driven by partially folded intermediates - incomplete structures that become fulfilled intermolecularly -favored by mild denaturation (i.e. acidic pH, but not strong denaturants like 8M urea) -mutations that destabilize native protein promote amyloid -in some cases, amyloid can be prevented by things that stabilize native protein (i.e. mutations, ligand binding)

folding funnel model

-top of funnel represents denatured conformations (max conformational entropy) -reason why protein eventually goes back to native state -lowest energy state is native state and protein will try to get to that -misfolded proteins can get "stuck" in those little dips on the side -denatured state has higher entropy -native state has lower entropy, will spontaneously occur

cooperativity of Hb

-transition from deoxyhemoglobin (T state) to oxyhemoglobin (R state) occurs upon O2 binding -iron ion moves into plane of heme when O2 binds -when O2 binds to iron, iron becomes slightly smaller which leads to movement -proximal histidine (component of alpha helix) moves w iron -resulting structural change is communicated to other subunits so that the 2 alpha-beta dimers rotate w respect to each other, resulting in formation of R state

hemoglobin

-transports O2, CO2, H+ -important role in pH balance -tetramer -2 alpha subunits of 141 residues, 2 beta subunits of 146 residues -4 AA chains (proteins) which are associated together (has quaternary structure)

polar R groups (hydrophilic)

-uncharged -polar, acidic (negative charge, COO-) -polar, basic (positive charge, NH3+)

polar

-unequal sharing -asymm distribution -bond involves EN atom (O>N>S>C>P>H) -e.g. water

denaturation

-unfolded -loss of secondary and higher order structure -noncovalent interactions are disrupted, peptide bonds aren't (AAs are still bound together) -results in loss of activity (bc structure = function) -can happen thru denaturing agents (e.g. urea, heat, pH) -e.g. RNase experiment

polarity and H bonding

-water has electric dipole -covalent bond between H and O where electrons aren't shared equally (cluster more w O)

why salts are soluble in water

-water has high dielectric constant (high value indicates greater ability to dissolve salts) -water is also polar and can disrupt ionic interactions -w/in salt crystals when you boil salt water, there are tiny amounts of water

importance of noncovalent interactions

-weak and transient -> gives macromolecules flexibility -provides stability to macromolecular structures (large # present in molecule, unlikely that all will be broken at same time) -essential to specificity and catalytic efficiency of enzymes

Henderson-Hasselbalch and buffers

-when [HA]=[A-], acid is 50% dissociated and pH=Pka -> this is point of max buffering capacity -slope is inversely related to buffering capacity -sum of buffer components doesn't change, only ratio does -sum of acid and conj base doesn't change, the ratio does -pKa relative to pH of solution and conc of buffer components determine effectiveness of buffers

tertiary structure

-whole structure of protein (almost always stabilized by hydrophobic core) -importance - AAs far apart in primary structure are brought closer and interact via noncovalent interactions between R groups -role of hydrophobic effect - nonpolar AAs in interior of structure, away from water -most proteins have combo of secondary structure motifs that fold to give protein its 3D shape