Biochemistry Exam 2

trypsin 

• endopeptidase

• serine protease = recog Rn-1 (amino that contrib carbonyl C)

• most specific

  • recog pos charged residues: Arg, Lys

• does not work w Pro (induces kink so cannot cleave)

chymotrypsin

• endopeptidase

• serine protease = recog Rn-1 (amino that contrib carbonyl C)

• recog bulky hydrophob residues: Phe, Tryp, Tyr (aromatic side chain)

• does not work for Pro

pepsin

• endopeptidase

• aspartic protease = recog Rn (amino that contrib NH))

• unspecific: Leu, Phe, Trp, Tyr

• does not work with Pro

amide bond hydrolysis: acid mediated mechanism

• for aspartic protease

• not catalyzed event = reactant not regenerated

• includes electrophile & nuc attack

amide bond hydrolysis: base mediated mech

• for serine proteases

• OH not regenerated

catalytic triad

• Asp - His - Ser

• always conserved in active site of enzyme of serine protease

• uses low barrier H to enhance nucleophilicity/ base characteristics → leads to easier breakdown via nuc attack

• acyl-enzyme intermediate = molec cov modified

cynanogen bromide (CNBr)

• cleaves peptide bond (internally)

• recog Met (high specificity)

• analogous to serine protease (endo)

• end w/ modified residue: homoserine

  • C-term not free

  • unlike ser prot that no mess w/ polypep chem

determining primary struc of prot

• reduction of disulfide bonds to sep polypep chain

  • oxidation of disulfide bonds w/ performic acid = irreversible w/ no cap

• break down polypep chain w/ two dif methods to gen dif set of peptide frag

  • ex. trypsin + CNBr (both v spec)

• determine seq of each frag w/ edman degradation

• use overlapping seq in each pep frag to determine seq of each polypep

• repeat frag w/o breaking disulfide bonds so can detect Cys

edman degradation

• used to sequence oligopep frags

• acid hydrolysis of PTC polypep yields PTH amino acid & intact polypep minus 1 amino acid from N-term

• one by one cleave from n-term and seq amino

• tag n-term w/ dansyl

reductive cleavage and alkylation of disulfide bonds

• oxidative process = reduce prot of interest & oxidize reducing agent

• expose to electrophile (iodoacetate) & sulfur attacks to prevent auto oxidation and reformation of disulfide bonds

  • this leads to capping of S

merrifield synthesis process

• use boc group to protect n-term & protecting group to protect side chain of amino acid of interest

• couple resin w/ fxnal group to boc+aa+protecting

• connects desired aa to resin via free COOH group

• remove boc group to free amino group

• attach second aa w/ free COOH (but has boc group to protect n-term)

• continue w/ desired number of aa

• remove protecting groups & then resin via chromatography

• = mature oligopeptide

merrifield synthesis overview

• synthesize c-term to n-term

• used to chemically synthesize polypep in solid phase (on column)

• want to install aa on resin

• ensure go to completion!

  • if low yield = nested frag = hard to purify

• limitations: 100-150 aa

boc group

• = protecting group on side chains w/ specificity

• protects amino group

• used in solid phase peptide synth (merrifield)

• all have free COOH

linkage of 2 peptides chemically = native chem ligation

• activate c-term & turn into thioester (using cysteine residue on n-term)

• link via side chain to cysteine S

• end in peptide bond & regen cysteine residue

• two peptides could be made via merrifield

• first step = no change in energy

• second step = favorable

mass spectrometry (MALDI)

• MALDI = matrix-assisted laser desorption/ionization

• mass usually = molec weight = vap/ionize molec = too strong & can break polypep chain

• MALDI = get molec weight of polypep but gentle = keep prot intact during vap/ionize

  • can determine accuracy of synth prot if mass same as expected (count up aminos)

conjugated protein

prot prodiced via cov interactions

• ex. glycoprot, phosphoprot, hemoprot

analytical purification

small prot applied to small, compact column (ug, ng)

preparative purification

bench work = big prot (mg, g) = purify a lot of prot

salting out (NH4)2SO4 precipitation

• continuously increase concen of (NH4)2SO4 to keep precip out new prot

• crude = does not lead to homogenous prot → but get as purified as possible & cut down contaminating prot

membrane dialysis

• used to sep by molec weight = small and large molec

• use dif pore sizes w/ cutoffs (keep desired prot in bag and everything else in solution)

• used to desalt prot (use very small prot)

• crude = not completely pure

osmotic pressure

water wants to goes back into bag so concen of big molecs = same

(why we need to seal bag in membrane dialysis)

gel filtration / size-exclusion chromatography

• sep of prot based on dif in size (and secondary dep on shape)

• use size-exclusion resin w/ dif porosities (large v small holes)

• pack into column & load mixture

  • small molecs can penetrate beads = small elute slow

  • large molecs = excluded from column = big elute fast

residence time & band width

• as prot size decreases = elutes slower = on coulmn for longer = last peak

• small molecs = broader band = slower & greater diffusion

• but same vol so area under curve of each peak = same

exclusion limit

molecular mass of the smallest molecule unable to penetrate the pores (beads) of given gel

Vo

• void volume

• volume of solvent space surrounding the gel beads = determined experimentally by using large standard molec like blue dextran

Vx

volume occupied by gel beads

Vt

total bed vol (= Vx + Vo)

Ve

elution volume = volume of solvent required to elute a solute from the column

Ve/Vo

relative elution volume = indep of the size of the particular column used (used to compare elution behaviors)

SEC Ve/Vo plot

• plotting Ve/Vo against logM = linear standard curve

• use to determine mass of unknown prot

• exceptions due to shape (assume globular nature)

  • ex. fibrinogen = behaves like larger molec than actual size

ion-exchange chromatography

• uses stepwise elution

• cation exchange

  • resin: neg charged, mobile cations as exchangers

  • large net pos = tightly bind to column

  • large neg = cannot interact w/ resin → elute fast (“salt out”)

• ideal method = usually start so all have net pos charge at beginning of elution & then charge pI for gradual elution in order of increasing pI

ion-exchange resins

• can have both ion-exchange & gel filtration habits

  • size also matters in some cases

• size factors can lead to deviations in expected & predicted elution based on pI

elution profile

can predict elution based on pI but size factors can lead to deviations;’l;l

affinity chromatography

• most powerful method of purification

• effective resin must be used = hardest part

• build column by self = extra specificity towards desired protein

  • only desired prot binds ligand (after washing column)

  • other contaminants are eluted out

effective resin production

• covalently link ligand (amino group) to agarose gel using CNBr as an activator

• activates OH group in agarose gel

• amino group = spec to desired prot

derivatization of epoxy-activated agarose

• gel pre-activated as epoxide

• makes it easier to link & more reversible

  • link epoxide & ligand

• spacer arm = dist between struc of ag bead & functional group

• sterics important

  • if ligand too close to bead = sterically inaccessible & binding will not occur

purification of staphylococcal nuclease by affinity chromatography

• on agarose

• gel binds to nucleotides like amino acids

• resin is built w/ T-residue

  • modified t-ligand = so amino group is attached & can bind resin surface

• leads to very homogenous mixture

  • wash out excess prot = only desired prot bound

• able to access bound prot

  • after washing = elute w/ actual ligand = easily and cleanly remove protein

slab gel electrophoresis

• separation (rate of migration) based on differences in mass/charge ratios of proteins

• prot migrate from cathode to anode

• pH 9 buffer used

formation of cross-linked polyacrylamide gel for electrophoresis (PAGE)

• polymerization of acrylamide & N,N-methylenebisacrylamide

• use radical polymerization (not very structured)

  • can change ratio of acryl & N,N → changes porosity

    • thus changes % of cross-link

Ferguson plots

• log mobility over % gel

• free mobility at 0% gel

  • when size does not matter (only dependent on charge)

• as % gel increases = size matters

  • lose mobility

    • small particle, high charge = high mobility

    • big particle, low charge = low mobility (greatly impeded)

SDS-PAGE

• SDS = amphipathic

• SDS-treated proteins = identical mass:charge ratios

• separation is solely based on molecular mass

  • can allow us to estimate molec mass of unidentified prot using standards

• relationship between motility & mass is logarithmic

SDS-PAGE & multi-subunit proteins

• gives molec mass of prot subunits, not whole prot

  • each polypep chain will have own band

• = disrupts the non-cov bonds that hold quat strucs tg

ultracentrifugation

• sedimentation coefficients for some bio materials

  • vary from small (small prot) to big (mitochondria)

• fractionate subcellular organelle

• based on mass and shape secondarly

zonal ultracentrifugation

• uses preformed sucrose density gradient

• then add sample & spin at high speed (centrifugation)

• let settle & see fractionation (particles sediment)

  • slow v fast sedimenting components

• can then poke holes and sep each fraction

• use sedimentation coefficients to separate (mass based)

• okay for proteins!!!!

isopycnic ultracentrifugation

• sep mixtures according to density

• only involves fractionation of subcellular organelles

  • prot have same densities

  • no proteins only organelles

• initially homogenous mixture becomes gradient based in density (in relation to density of CsCl or Cs2SO4 solution)

isoelectric focusing

• create pH gradient using ampholyte soln incorporated in gel

• pH gradient established (neg→pos)

• protein added & migrate in column based on pI valued

isoelectric focusing of hemoglobins

• dif subunits of prot have dif absorbance

• dif pI = dif fractionation & good separation of bands

2-dimensional gel-eletrophoresis

• 1st dimension = isoelectric focusing

  • fractionation based on pI

  • prot fall in order of decreasing pI

• 2nd dimension = SDS-PAGE

  • based on molec weight

• = leads to much better & more complex sep of proteins

peptide bond UV absorption

• aromatic absorption at 280 nm

• all prot absorb around 200 nm

  • due to amide fxnal group

• not all prot behave similar in amide region of UV absorption

  • amide absorption bands are dif

  • dep on secondary bond to amide

CD (circular dichroism) spectra of polypep

• delta E = dif in circular polarized light (measure of chiral props exclusively)

• uses polarized light = can distinguish between random coil, alpha, and beta

  • 3 distinctly dif curves (standards)

  • can use to estimate % of each 2nd struc in protein

trans peptide (amide) configuration

• all atoms lie in same plane

• due to geometry around C+N (120 degree bond angles)

• C alpha and C alpha 180 from one another

• more stable

• favored by bigger R groups = less steric hindrance

  • so most prot trans

cis peptide (amide) configuration

• C alpha and C alpha closer tg

  • hindered by R groups

• C+N still 120 degree

• less used by prot except glycine (H)

• less stable

• when many Gly = mixture of trans & cis = unstable protein

peptide bonds on protein backbone

• rigid peptide bond = C-N

• rotatable phi = N-C alpha

• rotatable psi = C alpha - C

• can change config by rotating phi or psi (some more fav than others)

Ramachandran phi/psi plot for proteins

• shaded areas = very stable (low E) bond configs = highly populated in backbone

• white area = high energy, unfav configs

• not every combo fav = sterically inhibited

• axis = tortion angles

Ramachandran phi/psi plot for Gly

• more shaded areas than normal = many more fav conformations

• only have H side chain = no chiral center = less steric hindrance

• = more flexibility to amide and can adopt phi/psi configs that other amino acids cannot 

• = high mobility & highly disordered 2nd struc

alpha helices

• H bonds w/ carbonyl & N

• R groups pointing outward = easily accessible

• due to local folding

• residues = big number = # of residues per 360 rotation

• rise = overall rise

• small number = # of atoms in H-bonded ring (how many atoms sep O from H)

comparing 3₁₀ , 3.6₁₃ (alpha) , 4.4₁₆ (pi) helices

• 3₁₀ = 3 residues per turn

  • small cross-sectional area

  • thinner, stretched out

  • less amino acids

• 3.6₁₃ = 3.6 residues per turn

• 4.4₁₆ = fatter, contracted

• all have intra H-bonds holding H tg & R groups on outisde

  • so all same stabilization

analysis of alpha helix R-group composition

• extract piece of backbone of myoglobin

  • determine location of e-helix = R group analysis

    • prot recog R group orientation (on outside surface of helix)

dipole moments in helices

• fractional charges between neg c-term and pos n-term

• all peptide bonds orientated in same positions = dipole moment in helix

• can get electrostatic interactions

  • lead to fxnal role

  • ex. phosphate group binding

beta (pleated) sheet

• primary struc interacts to fold in on itself to align like beta sheet

• H bonds stabilize NH and carbonyl O

• R groups on top or bottom of sheet surface → determines fxnal properties

  • can make amphipathic by using differntly charged R-groups on each side

antiparallel beta sheet

• more stable = intra H-bonds more strongly aligned

• each carbonyl can be acceptor for 2-3 bonds

• one sheet N→C and one C←N

parallel beta sheet

• both in same orientation C←N

• intra H-bonds offset (not as perfectly aligned) = less stable

• non linear H-bond = weaker

beta sheet stacking

• most rigid structure = Ala & Gly

• very strong

• beta can easily stack (intercalations) (conformations of R groups highly complementary)

• H and methyl pack tightly

types of beta sheet connections

• hairpin = antiparallel (just need single turn)

  • stabilized by 1 H-bond

  • 2 aa in turn but use C alpha 1 and C alpha 2

  • alternate turn by rotating tortion angles

• out of plane crossovers = above & below plane (parallel)

  • more work / more complicated turn

gamma turns

• caused by proline = ring struc induces bend

  • limits geometry & introduces bend / kink

• C alpha 1 and C alpha 3 involved

omega loop

• backbone bending motif

• out of plane crossover of beta sheet

fibrous proteins

• insoluble

• usually comprised of 1 type of secondary struc

• types: alpha keratins & collagens

alpha keratin

• start as dimer

  • n-terminal head 

  • coiled coil rod = alpha helix wrapped tg

  • c-term tail

  • connected via hydrophobic interface (dissymmetric distribution of aa)

  • dimer interface due to hydrophob primary struc

• protofilament

  • head and tail more globular = can self associate to form protofilament

• microfibril = disulfide bonds lead to formation and stablization

collagen

• very strong fiber = used in connective tissue

• distinctive amino acid comp: Gly, Pro, Hyp

  • Hyl also present (involved in cross-linking & glycosylation w/ O)

• triple helical structure = 3 polypep chains wrapped around one another

• stabilized by H-bond at interface

  • Gly = contrib NH (donor)

  • Pro = contrib O (acceptor)

• glycosylated at Hyl residues w/ Glc-Gal post-translational

• covalently cross-links Lys, Hyl, and His side chains in collagen

x-ray diffraction

• image produced from x-ray scatters

• uses crystal of myoglobin (static state)

• this image = well-behaved = each prot molec in crystal is identical = good resolution

• compare multiple diffraction patterns = create 3D structure

• lower resolution = hard to determine structure

  • h-bonding = no diffraction so must rely on heavy atoms

  • must have good resolution so can differentiate between atoms

2D NOESY spectra

• must use 2D spectrometry to determine protein internuclear distances

  • 1D = too many overlapping signals

• use “off-diagonal” method to determine internuc dist of all protons ( r )

  • many distances = put into computer program = determines how prot must be folded to account for all internuc distances

• shows natural aq state behaviors

• inconsistent fitting (less overlap) = intrinsic disorder in dynamic prot or insufficient number of distances

helical wheel

• represents distribution of polar & nonpolar residues in helix

• helix = amphiphilic = one side np & one side polar

• inner facing proteins = hydrophob = hide from aq environment

• asymmetric primary struc of aa leads to folding of 2/3 structure

domain definition

• long peptide chain can fold into single peptide domain (like myoglobin)

• or single polypep chain can fold into 2 domains independent of one another

  • will fold same way or w/o presence of other = folding solely based on primary struc

• if can truncate polypep & one express one & still folds same = domain

• ex. immunoglobin

  • light & heavy chains

    • single polypep chains that fold into 4/2 indep domains

beta - alpha - beta

• super secondary structure

• parallel beta sheet

• uses helix as cross over

• stabilized by H bonds

beta - hairpin

• super secondary structure

• 2 anti parallel beta

• fold in on each other to produce hairpin turns

• stabilized by H bonds

alpha - alpha

• super secondary structure

• alpha and alpha separated by turn

• stabilized by dissymmetric distrib of hydrophob/phil interactions at interface 

  • like alpha-keratin

greek key motif

• super secondary structure

• series of antiparallel beta due to folding

• create new interactions from folding

beta barrel

• super secondary structure

• 2 greek key motifs

• cylindrical → pairing introduces pore/hole

• found in memb-bound proteins where hole is needed to allow things to pass thru memb (transport protein)

Cro dimer

• dimer that binds DNA duplex

• dimer = fxnal form

  • monomers cannot bind DNA duplex

• H bonds hold each monomer tg

• dimerization stabilized by antiparallel beta sheet interface

• protruding R groups on alpha helix available to perfectly penetrate into major & minor groove of DNA

  • stabilized w/ H-bonding

zinc finger protein

• combination of beta sheet, alpha helix, and random coil

• Cys-Cys-His-His family

  • 2 His = imidazole ring

  • 2 Cys = S

• Zn anchors structures and forces fold

• very strong ability to bind protein → often attached to DNA

• when multiple zinc fingers bound tg = at least one able to bind to DNA

  • via alpha helix and protruding R groups w/ specificity to bind grooves

leucine zipper

• dimer bound to DNA

• 2 alpha helix bound tg by Leu-Leu interface

  • not hydrophob residues!!!

• protruding R-groups bind to DNA

structural hierarchy in proteins

• primary (amino seq in polypep chain)

• secondary structure (helix)

• tertiary structure (polypep chain folding into 3D struc)

• quaternary structure (four sep chains associating thru non-cov interactions

quaternary structure (hemoglobin)

• 2 types of chains: 2 alpha & 2 beta

• fxnal form = due to order of association

• associated thru noncov interactions (very weak so easily disrupted)

• hemoglobin = O2 binding prot

  • binding to one site in one domain effects entire molec

  • binding = leads to struc change = changes affinity of all other sites

  • = cooperativity (communication)

• differs from single domain in 3 struc (myoglobin) = independent binding (O2 binding one sit does not effect others)

chemical cross-linking of oligomeric proteins

• start w/ 2 subunits of quat prot & dimethylsuberimidate or glutaraldehyde

• Lys on subunits reacts & creates cov bridge / cross-link between subunits/monomers

• allows quat prot to undergo SDS or native so can properly determine molecular weight of proteins

  • SDS denatures and breaks down non-cov bonds that normally hold quat struct tg

  • get multiple bands (one per secondary struc)

• limitations: concentration important

  • too much = cross linking of other dimers = create new, unwanted tetramers

protein-protein interactions

1. surface-string

   1. primary struc of 1 subunit inserts self into another subunit

2. helix-helix

   1. 2 polypeps react to form helical interface

3. surface-surface

   1. 1 surface complementary to other = topology allows perfect fit/binding of interfaces

   2. most common

induced fit model

• free form (random coil) induced to change conf to adopt defined secondary struc upon binding 

• shows plasticity of geometry

• leads to stronger binding upon fold

GRASP

• graphical representation and analysis of surface properties

• uses colors to determine distributions of ionic charge

  • ex. red = neg, blue = pos, white = neutral

• can use to predict how prot will interact w/ other charged molecs

• computer-derived molec

Chou-Fasman method

• set of rules that allows you to determine what secondary structure will arise based on characteristics of primary structure/amino acid composition

• uses frequency and propensity

• input primary struc into computer = determine where alpha, beta, and turns will occur based on polypep chain & propensity & hydropathy values

frequency

• the statistically derived likelihood or propensity that a specific amino acid will be found in a particular type of protein secondary structure

• greater frequency = greater likelihood amino acid will contribute to forming structure 

• = f alpha = n alpha / n

  • n alpha = number of amin acid residues of the given type (ex. alanine) that occur in alpha helices

  • n = total number of residues in of this type (ex. total number of alanines in protein set)

propensity

• the tendency of an amino acid to prefer one type of amino acid structure over another

• determined by: P alpha = f alpha / <falpha>

  • <falpha> = average value of f alpha for all 20 residues 

• when P alpha > 1 = residue occurs w/ greater than average frequency in an alpha helix

  • 1 = average frequency

  • if p very low = strong break = usually Proline or Glycine (induces kink & disrupts secondary structure)

• changes over time as more prot analyzed = living study

Rose method

• occur on surface of protein

• occur at positions along a polypep chain when hydropathy goes from max to min (excluding helical regions)

• predicts where turns in struc will occur

hydropathy scale

• very high positive values = very hydrophobic (interior)

• very negative = very hydrophilic (exterior)

reductive denaturation & oxidative renaturation

• use reducing agent (mercaptoethanol) & denaturing agent (urea) to break down tertiary and secondary struc = become random coil

  • reduction = remove disulfide bonds (S-S to SH)

  • denature = unravel (cystine → cysteine)

• remove both at once = back to same (FOR RNase and some prot)

• remove just reducing agent = disulfide bonds form in wrong places

  • then remove urea = enzymatically inactive prot generated

  • have to add small amt of reducing back to loosen bond just enough for port to hopefully refold into correct protein

heat-induced protein denaturation curve (for RNase A)

• plot fractional change against Tm

  • fractional change = any measured parameter

• S-shaped curve = implies cooperativity

  • once begins denaturing at one site = everything begins to denature

  • steeper slope = greater cooperativity

• denaturing & renaturing = reversible process

  • due to temp (denature w/ heat & renature upon cooling)

  • conclusion: everything polypep needs to fold is in primary struc

    • catalytically active after each transition thru curve

Tm

• melting temp for proteins

• characteristic value

• measures thermal stability of protein

• higher Tm = harder to unravel = more stable prot

Hofmeister series

• shows the effect of concentration and slats on Tm

• chaotropic salts = reduce Tm & destabilize protein

  • make easier to denature & unravel prot by weakening hydrophob bonds

some determinants of protein folding

• helices/sheets predominate in proteins bc they fill space efficiently

  • why prot density = same

  • secondary struc pack very efficiently

• prot folding is directed mainly by interna; residues (protein folding is driven by hydrophobic forces → the hydrophobic effect)

  • hydrophob must be folded in core

  • immobile H2O molecs in unfolded prot in core → more mobile in aq

  • increases S = neg G

• prot folding dep on primary struc (amino acid sequence)

  • but not case for all prot → may have other influencing factors

    • slow rate of folding = post-trans folding

    • fast rate of folding = folding during trans (on ribosome)

hydrophobic effect & protein folding

• ex. fav folding for hydrophob molec moving from water to hydrophob solvent

• H = post

• S = neg

  • water molec release from hydrophob molec = increase entropy

• = -G

  • fav to fold (denature prot to folded prot)

Levinthal Paradox

• crude estimate of the time required for protein folding

• conclusion: proteins must fold via ordered pathway or set of pathways bc time for random folding = greater than age of universe

  • based on conformations of phi and psi (not based on Ramachandran bc unfold prot)

stopped-flow device

• very fast way to mix two solutions (very low dead time)

  • ex. reducing & dentauring agent

• then observe prot folding in detector

  • denat/renat or earlu stages of prot folding

• vary dead times to observe dif stages of prot folding

• detection/analysis via:

  • UV absob (look at aromatic aa = primary struc)

  • CD spectrum (measure secondary struc) = better method