bibc 100

fours types of weak interactions between water and biomolecules

hydrogen bonds

ionic interactions

phosphorylated aa

serine, threonine, tyrosine

histidine

• regulated enzyme activity

• protein/protein interactions

• protein degradation

• signal transduction

sulfanated aa

tyrosine

• hormone receptor-ligand interactions

• G protein signaling

Methylation

lysine, arginine

• epigenetic regulation

• histone function (charge is maintained)

Hydroxylation of aa

Proline, lysine

• O-linked glycosylation

• phosphorylation

Carboxylation

glutamate

• affinity for Calcium ions

hydrogen bonds

between H2O due to electron structure across O-H

O more electronegative

electrons not equally distributed → dipole

due to tetrahedral arrangement of orbitals → interacts 4 neighbors

hold h-bonds with average of 3.4 water molecules

O-H h-bond length x2 the covalent

• dissociation E is 23kJ/mol Hbond < 470kJ/mol covalent

• lifetime between each other is 1 to 20 picoseconds

high cohesion → melting/boiling point are higher for water cus stick together

polarity and its affects on interactions

polar → large electronegative diff

• all weak interactions except hydrophobic

non-polar → small electronegative diff

• hydrophobic and van der Waals

amphipathic → part hydrophobic part hydrophilic (phenylalanine + phospholipids)

• all weak interactions

ionic interactions

electrostatic interactions based on O-H bond dipole of water → interacts with chaarged ions

ionic bonds are tranfered interactions are not

hdipole+ with Cl- → attractive

ionic salts want to be disordered and not in Crystal structure

hydrophobic interactions

free moving water is favorable over caged water

less total surface area → smaller decrease in entropy → more energetically favorable

• maximum possible entropy means more energetically favorable

• aggregation

hydrophobic interactions do not occur because non-polar solutes attract one another

van de Waals interactions

very weak transient version of other electrostatic interactions

• fluctuating dipole

random variations in electron positions around a nucleus causes transient dipole

distance is important

• equilibrium is achieved when attractions and repulsion are balanced

lipid bilayers are a result of ___ effect on ___ solutes

hydrophobic + amphipathic

polar head on the amphipathic molecule

causes what look like ordered structures

__ molecular interactions drive protein folding

weak

hydrophobic inside hydrophilic outside

charged/polar amino acids at the surface to participate in electrostatic interactions

release of ___ water facilitates formation of___ complexes

ordered + enzyme-substrate

non polar solutes cause water to be constrained into more orderly orientations along interface

polar cause constrained water molecular motion to lesser degree bc compensatory electrostatic interactions

disruptions of ordered water drives interaction of polar substrates with the complementary polar surface of an enzyme

• entropy increases as the water molecules associated with the substrate and enzyme are displaced

__ interactions are critical to __

weak + biomolecule stability

cumulative effects of interactions has significant strength

large # of weak interactions → more stable complex

to dissociate 2 biomolecules joined through multiple weak interactions → need disruption all at the same time

• this is less probable so the stability of the weak interactions is greater than would be expected if they only had an additive effective

proteinogenic amino acid

alpha carbon - carboxyl and amino group bonded to same carbon

stereoisomers

L AA → amino group before carboxyl clockwise

D AA → carboxyl before amino clockwise

20 AA in 5 groups based on side chains

nonpolar or aliphatic (hydrophobic)

aromatic (varies)

polar uncharged (hydrophilic)

positive charged (basic)

negatively charged (acidic)

stacking of aromatic AA

pi-pi stacking

• stabilizing protein structure

• mediating interactions with other protein, DNA/RNA, small molecules

overlapping orbitals

interaction between TRP side chain in DNA polymerase eta subunit and adenine residue in DNA

positive charged AA

lysine

• primary amino group , pka = 10.53

arginine

• guanidinium group , pka = 12.48

histidine

• aromatic imidazole , pka = 6

lysine and arginine stabilize protein binding to nucleic acids interacting with __ backbone

phosphate

positive charge on Lys/Arg with negative phosphate groups on backbones

involved in protein interactions w nucleic acids

ex: p53 DNA binding protein contacts the DNA backbone through K120, R248, R273 and R280

Mutations of these residues in p53 often result in cancer due to disrupted p53 function

histidine is — and — to small shifts in —

aromatic and sensitive to small shifts in pH

aromatic → charge is shared across bonds

only ionizable amino acid with a side chain pka near physiological pH

small shifts in pH can alter charge

common enzyme active sites and participates in many catalysis reactions

negatively charged AA side chains

acidic

found at the external face of proteins where they participate in electrostatic interactions

(-) charge of asp/glu complementary to the charge of lys/arg can mediate protein interactions

asp/gly involed in coordination of metal ions

general pkas of side chain

lys/arg → 10-12

his → 6

asp/glu →3/4

Two other amino acids have been found to be genetically encoded and incorporated into proteins

selenocysteine

• TGA-codon

• pka = 5.2

cysteine with sleneium instead of sulfur

pyrrolysine

• found in methanogenic archaea and bacteria

lysine with pyrroline ring attached

bc selenocysteine is an effective — it is important in the active site of many —

nucleophile + redox enzymes

selenium is more efficient nucleophilic attack

ex: required active site of gluthione peroxidase (antioxdant protect cell’s lipids)

seleneocysteine sec is encoded by an —- —- —- —- in some proteins

opal stop codom UGA

to encorpoart sec into proteind uring translation mRNA transcirpt must contain 2 structural elemetns

1. opan stop codon complementary to tRNA carrying sec

2. secondary structural element in 3’ untrans region of mRNA formed by SECIS

60 nucleotide in length & forms stem loop

secondary structure of secis

The translational machinery pauses at the opal stop codon

The stem-loop structure of the SECIS physically interacts with the translational machinery allowing the Sec tRNA to be recruited to the mRNA being translated

The Sec tRNA delivers Sec to the opal stop codon

The 10 bp tRNA acceptor stem of Sec tRNA is longer than most (most are 7-9 bp) to facilitate interaction with the paused translational machinery

pyrrolysine

present in active site of methyl transferase

• ring coordinates positioning and displaying the methyl group

incroporated at a stop codon Amber

zwitterions

dipolar ions acts as acid or base

at neurtral pH → aa are present as zwitterions

pk1 = dissociate of H+ from carboxylic acid group

pk2 = amino group

titration curve for glycine

2 buffering areas

dissociation of H+ CA is pk1 = 2.3

isoelectric point: 5.97 (charge is 0)

dissocaition from amino acid = 9.6

structure of aa affect pka

pka of carboxyl is lower than other CO

→ opposite charge amino group stabilizes the zwitterion

pka of amino is lower

→ electronegative carboxyl pulls e- from amino group

titration for charged aa

2 zone of buffering + zone of buffering for loss of H+ for side chain

histidine = 6

lys/arg = 10-12

asp/glu = 3-4

peptide form through condensation

joined through amide group and hydroxyl

amino acids in peptide chain are called residues

where/what are peptide chains

composed of repeating amino nitrogen, alpha carbon, and carbonyl

have amino and carboxyl terminus

• oligopeptide: >2 residues

• polypeptide: <10,000 kiloDaltons

• protein >10,000

examples of peptides

small are important

• aspartame, oxytocin, amantin (toxin from mushrooms inhibits RNA polymerase

aa side chain can coordinate metal cofactors that enhance enzyme activity

aa: his, cys, asp, glu, selenocysteine

metals: iron, zinc, magnesium, calcium

ex: rebonucleotide reductase → iron atoms coordinated in the active sire by glu, his, asp

• triggers free radical production in tyr to deoxygenate ribonucleotides

alcohol dehydrogenase

• zinc atom is coordinated in active side by cys and his residues

• zinc binds an alcohol mol in position to facilitate hydride transfer to coenzyme nicotinamide

oxygen binding proteins contain — — groups to bind to oxygen gas

heme prosthetic

none of aa side chaine in proteina re suitable for reversible binding oxygen

• solutions: coordination of heme prosthetic groups containing iron to reversibly bind oxygen

modifcations to aa

add/remove charge groups

add functional groups

local changes → protein structure → leading to changes in protein function

• enzymatic function

• protein localization in cell

• ability to form complexes

post transitional modifications

phosphorylation (ser, thr, tyr - eukaryotes) (his prokaryotes)

sulfation - tyr

methylation (lys, arg)

phosphorylation importance

transcirptional activity

proetin stabilty

subcellualr localization

creat confomartipnal change -> ATP

hydroxylation

proline and lysine

permit other modifications such as O-linked glycosylation or phosphorylation

carboxylation

glutamate for clotting

increases affinity for Ca+

glycosylation (linked)

O-linked: to hydroxyl oxygen of Ser, Thr, Tyr, Hydroxyproline, hydroxylysine

N-linked: to nitrogen of Asn (or very rarely to Arg, only known in bacteria)

C-linked: very rare, to Trp

critical to protein stability, at least 40 human diseases are a result of mis-glycosylation, none of which have a known cure!

acetylation

lys

epigenetic regulation

histone structure

interaction with DNA

antibody glycosylation

stabilizes them

AB for immunity

• directly block viral entry into cells

• AB tagged molecules or organism recognized by other immune cells

— groups can be attached to amino acids

to facilitate —-

hydrophobic + protein localization to membranes

Myristoylation: Myristic acid (C14) tail covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue, important for signaling and apoptosis

Palmitoylation: covalent attachment of palmitic acid (C16), to cysteine and less frequently to serine and threonine residues of proteins. Can be reversibly removed, important for hemagglutinin-mediated influenza infection

Prenylation: transfer of either a farnesyl or a geranyl-geranyl moiety to C-terminal cysteine(s) of the target protein, important for protein-protein interactions

Hydrophobic groups can be attached to amino acids to facilitate protein localization to membranes

Hydrophobic groups insert into the cell membranes – this allows proteins to localize within the cytosol to the face of specific membranes

Palmitoylation is readily reversible, and can regulate function of a protein through changes in localization

i.e. Some signaling proteins are not functional unless they are present at the plasma membrane, and their function can be negatively regulated by removing them from the membrane (depalmitoylation)

— acetylation is important for regulation

of DNA accessibility and gene transcription

Lysine

DNA is packed around histone proteins in our cells

Histone proteins are spherical-shaped octamers made from 5

types of subunits, named H1-H5

DNA is wrapped around the exterior of the histone octamer

Lysine acetylation weakens histone interactions with DNA

positive lysine residue interact wiht negative phophaste on DNA

K64 can be acetylated which weaken interaction with DNA cus there is less charge

DNA falls off histone

disruption of electrostatic interaction

loosed DNA → opens up interact w transcriptional machinery

regulate whether genes are turned on or off

chracteristic of protein

size, charge, polarity

chromatography → protein run through column with beads/matrix

separate from one another depending on structure

• charge → ion exchange chromatography

  • beads have negative charge + proteins that are negative move fast

  • positive take the longest

  • anion change is the opposite

• size → gel filtration

  • beads w channels → large protein goes fast

  • small protein go slow

• polarity → hydrophobic interaction (Reversed phase)

  • hydrophobicity

  • nonpolar stationary phase

acivity assay

try to find specific protein

sequence multple seperations

peptide bonds and rotation

dipole and bond is shorter and rigid

no fre rotation around bond

phi (ϕ) Rotation is around the N-Cα bond

psi (ψ) Rotation is around the Cα-C bond

large side chain aa have fewer possible angles of rotation

phi psi angles plot

alanine

phi psi angles plot

general

• glycine

• Proline

• Before proline in the peptide chain

phi psi angles plot

glycine.

phi psi angles plot

proline

right handed alpha helix, beta sheets, left ahnded beta sheets

alpha helices

stable bc hydrogen bonds

• H attached to N

• electronegative carbonyl O attached to peptide bond C

net dipole : amino has partial positive, carboxyl has negative

right handed are more stable for L amino acids

less likely to find glycine and proline in a helix

stability effected by:

• phi/psi bond angle

• proximal r groups (opp charges stabilize)

• bulky R group destabilize

• gly too much rotation, pro no enough

• same aa can destabilize

helical wheel projection

Amphipathic helix

(one side polar, one side nonpolar)

one side of the helix exposed and one side packed against the interior of the protein

Nonpolar residues - interior protein, hydrophobic

polar residues - helix is hydrophilic - fully exposed

HGH is amphipathic helices

beta shrands

antiparallel - amino to carboxyl, next is carboxyl amino

• most stable - hydrogen bonds

parallel - carboxyl to amino for all

• carbonyl dont match up as easily, not 180, less stable

beta barrel

common

hydrophilic exterior, hydrophobic

each strand has one face in our face out

alpha/beta structure

repeats of alpha beta strand

frequent domains

ex: leucine rich repeats

inner beta sheet, out alpha helices

alpha+beta

any combination of a and b

SH2 domain twisted beta flanked by alpha helices

coiled-coil

• long alpha helices intertwined between each other

• strong and flexible

• 3.5 aa per turn not 3.6

• repeat period of 7 aa - heptad repeat

  • hydrophobic interactions stabilize

  • salt bridge can stabilize ionic electrostatic interaction

keratin

protonfilament: 2 cc in staggered fashion (4 h)

protofibril: 2 staggered protonfilament (4cc, 8 ah)

filament: 4 protonfibrils twisted in right hand conformation (16cc, 32 ah)

keratin stabilized by

• helices bundled in staggered conformation

• disulfide bonds btwn protofilament and filament

collagen

repeats of pro-hyp-gly

loose left handed helix

triple helix bundled

can hbond to each other

staggered super helices

fibroin

ala and gly

tightly packed

hydrophobic side of each stack against one another

crystalline beta sheets of fibroin are interspersed with amorphous regions

• amorphous is what can stretch to allow for flexibility

beta barrel and carrier protein

retinol binding protien

• has a hydrophobic cavity where VA can go to where its needed

green fluorescent protein

• protects against aqueous environment

• gfp

• ef hands

• antigens

• retinol binding

• rubisco

enzyme active sites

beta propeller

• 4 beta strand twisted

• neuraminidase: flu virus protein

  • uses hemagglutinin HA to being to receptor protein glycosylated with sialic acid

  • active site made by loops stuck to beta strands (kinda flexible is important)

TIM structure

• stable core barrel strucutre

• rubisco: catalyzes carboxylation

  • active sire on top between loops

bind DNA and calcium

ef hand long loop - binds calcium

short - binds DNA

• charged polar residues asp and glu

CDPKs: conformational change after Ca binds to EF hands

• important for signal transduction

• allows for site to be open

diseases like malaria causes it → block activation of the kinase so it cant get it

antigen binding on AB

beta barrels bind to the antigens

loops bind the antigen

bind to organism for them to be degraded

toll-like receptors (TLR) are embeded into extracellular membranes

TLRs ar the security guards of immune system

made of the leucine rich

prediction of tertiary structure: levinthal’s paradox

in vivo porteinf old in 10^-1 → 10^-3 sec >98 orders

folding is too fast to be random

folding is due to collapse into most thermodynamically stable state

native protein vs denatured

folded - native (weak IM interactions maximized)

1. Native is tightly packed, a compact hydrophobic core

2. Determined by the amino acid sequence and how it interacts with the aqueous environment

3. Each protein sequence yields a unique native structure

4. The native folding state is thermodynamically stable (it represents the lowest achievable energy state for that protein)

unfolded - denatured (structure open side chains loosened)

step 1 of protein folding

hydrophobic collapse forms molten globule

into semi molten state

reduce in # protein conformations possible

peptide backbones hydrogen bond w one another and alpha helices and beta sheets emerge while protein collapse

unfolded → bury core (hydrophobic effect) → 2° → molten globule (loose 3° structure)→ 3° → 4°

molten globules & native fold

stable

next step is slower → arrangment of all side chains

lower free energy not as low as native fold

native fold:

• weak IM interactions w aa are maximized

• released energy w bonds → this more thermodynamically stable

thermodymics

∆G = ∆H - T∆S

change in free energy = change in enthalpy - change in entropy

∆G is negative → E is released more spontaneous

large ∆S → more spontaneous decrease in entropy

smaller ∆H → more spontaneous

enthalpy for folding

folded (native) → low enthalpy/entropy

unfolded (denatured) → high enthalpy/entropy

system w folding → decrease enthalpy, increased entropy

energy funnel diagram

troughs represent intermediates

wide funnel → lots of folding paths

anfinsens

1. ribonuclease a (degrades RNA) treated with B-mercapethanol and urea to denature protein

2. urea removed and reformation of Hbonds occur 1% activity regain

3. b-mercapethanol removed reform disulfide bonds 100% activity

denaturation

took active native folded enzymes and disrupt them

denature

• pH change - alter ionation of aaa side chains disrupts Hbonds

• heat - add E

• detergents - amphipathic molecules associate with nonpolar residues

• chaotropic agent - ion/small organic molecule at high conc. disturb IM interactions

  • Urea - interferes with H bonding

  • Beta-mercaptoethanol breaks disulfide bonds between cysteine

problems to folding

aggregation

non native disulfide bonds

isomerization of proline

removal of misfolded proteins

endoplasmic reticulum

Partially folded or misfolded →

• PDI, PPI, HSP70, HSP 10/60 chaperones: assist folding

• 26S Proteosome: degrade misfolded protein

Misfolded and aggregation

• IRE1 signaling in unfolded protein response (trigger cell death)

• HSP100 break up aggregate

Protein Disulfide Isomerase (PDI)

Non-native disulfide bridge formation

hydrophobic inside of PDI

serve as point of contact for unfolded protein

oxidized or reduced forms

• oxidize helps forms disulfide bonds

• reduced PDI helps break and reform

Peptidyl Prolyl Isomerases (PPI)

proline is only one in cis conformation (trans 4x stable)

PPI switch proline form cis to trans

immunophilin recognize substrates through 4 aa motif

• beta barrels

parvulins (PIN1)

target substrates proteins where serine or threonine precede pro

only recognizes substrates after phosphorylation

• too much PIN1 is around, these proteins are more readily activated and sometimes active when they shouldn’t be

HSP 60/10

GroEL cylinder

unfoled protein in one half and then the other

HSP10 (GroES) is the lid

Parts:

• Apical (alpha beta motif)

  • forms opening whern unfoled enters

  • flexible

  • hydrophobic

• intermediate

  • allow ATP/ADP diffusions

  • flexible hinges

• Equatorial

  • ATP bindings

  • stabilise chambers

• GroES: 7 subunit structures

  • β sheet

  • β hairpin (roof)

  • Mobile loop (interacts with GroEL)

hsp 70

nucleotide binding domain - binds ATP

substrate binding domain - clamping to substrate binding domain

closing and opening domain when ADP→ATP

• take on a new substrate

hsp 100

detangles misfolded protein

each subunit has ATP bound which helps tug misfolded protein through

how does accumulation of misfolded proteins get recognized to undergo cell death

ER-localized HSP70 called BIP

PERK1 kinase that stops translation of new proteins

ATF6 contains transciption factor that is released to turn on genes to help clean up

• golgi

IRE1

• causes splicing of mRNA transcript that activates expression of genes needed for the UPR

• causes degradation of most other mRNA transcripts

• induces apoptosis if it remains active too long Iif BIP does not re-associate)

How do aggregated and misfolded proteins get degraded?

1. If they can be disaggregated they are degraded by the 26S proteasome

2. If they remain as an aggregate they are disposed of through autophagy

misfolded go to cytoplasms through

• ubiquitylated (targets them to 26S for degradation)

  • small lysine residues

disease and folidng

form very small stable beta structures

• amyloid beta - alzeimers

• tau protein - parkisons

native fold can misfold into beta strands

• large aggregated beta sheets