BCH210 midterm

Covalent bond vs. non-covalent bonds in terms of structure

Covalent: hold together amino acids

Non-covalent: allow chains to fold into final structure

Protein cofactors

non-protein molecules and metal ions that assist in structure and/or function

Metal ion cofactors

may interact with the protein or be involved in enzyme catalysis (Ca2+, Co+, Mg2+, Zn2+)

Prosthetic groups

typically larger structures; tightly bound molecules by covalent or non-covalent forces

Transporters: covalently or non-covalently?

Non-covalently (ex: O2 binding to heme)

Cofactors: covalently or non-covalently?

Both

Coenzymes

serve as "shuttles" for commonly used functional groups in chemical reactions, so the reaction can occur properly

Chainbow image of protein

can visualize in 3D, backwards from blue to red (blue is N-terminus, red is C-terminus)

Different types of interactions (5)

1. Covalent forces

2. Ionic/electrostatic/salt bridges

3. Hydrogen bonding

4. Hydrophobic interactions/effect

5. VDW

Covalent forces

- strongest

- depends on electron sharing

- nuclei closes together

Ionic/electrostatic interactions

- 2nd strongest

- strength depends on polarity of charged species

- aka salt bridge: full charges

Hydrogen bonds

- 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

- depends on entropy of water being released, causing hydrophobic regions to come together

- hydrophobic effect

VDW

- weakest

- non-covalent

- depends son size of atoms, distance between them

Why is water an important molecule?

- can form up to 4 transient HB (unequal sharing of electrons)

- important for molecule stabilization

- important for formation of complex structures (can bring distant parts of molecule together)

Water and biochemical molecules

- water can participate in hydrolysis reactions (h20 breaks amide and carbonyl bond)

- macromolecules may fold to exclude water -- preventing reactions from occurring

- maximizing number of interactions of hydrophilic functional groups with water for solubility

Biochemistry and drug design

Binding interactions are IMF are important for drug design -- presence or absence of functional groups can assist with solubility and binding to specific targets

WEEK 2

structure of amino acid

amino group + carboxyl group +hydrogen group + R group

what is an essential amino acid

body cannot make it, must be obtained from our diet - depends on # steps it takes to mak

chiral amino acids

most are chiral, with exception of glycine

which chirality isomer is more physiologically relevant?

L isomer

how does chirality arise?

when all functional groups on alpha carbon are different

zwitterion

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?

BJOUXZ

amino acids can be metabolized to form what other important molecules?

- hormones

- neurotransmitters

- DNA/RNA

- energy-producing intermediates

Disulfides bonds

- covalent interactions

- can be intrachain or interchain

- stabilize structures

- PDI enzymes help catalyze this oxidation reaction

secreted vs cytosolic proteins: disulfide bonds

- 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?

reducing agents in cytosol

post-translational modifications (5 groups)

1. disulfide bond formation (stabilization)

2. phosphorylation (stabilization, increase solubility)

3. ubiquitination (degradation)

4. glycosylation (signaling)

5. methyl, acetyl, hydroxyl, carboxyl additions

GFP: aa modification

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

amino acid segments are properties conserved for structure and/or function

pH

-log[H+]

at lower pH values meaning?

more H+ present

what does pKA measure

strength of an acid

low pKa?

low pKa = higher Ka

stronger acid

choosing buffers around what range

+/- 1 pH unit around pKa

choose pKa closest to pH you require at given temperature

what is the point of buffers

resist changes in pH so certain functional groups will be maintained (maintain protein structure/function)

isoelectronic point (pI)

pH when charge of molecule is 0

how to calculate pI

1. see where all functional groups protonate/deprotonate

2. line up in order

3. go up and see when overall charge goes from -1 to 0 to 1

4. average those values

when pKa > pH

protonated state (think about NH3+ instead of NH2 at ph 7.4)

Bohr effect

- at lower pH, His146 is protonated and creates salt bridge to Asp94

- favors deoxygenated structure of hemoglobin

Blood's buffering ability

bicarbonate is main buffering species -- can accept and donate H+ to prevent changes in pH

with h2o, co2

when pH > pKa

deprotonated state (think about COO- instead of COOH in amino acid at physiological pH 7.4)

physiological pH

7.4

WEEK 3

Primary sequence of protein

linear sequence of amino acids

Primary sequence directionality

- amino acids joined enzymatically in condensation reaction

- N-terminus side is start of amino acid chain

- peptide bond goes through alpha carbons

Peptide bonds = planar

- peptide bonds are polar but uncharged

- peptide bonds have partial double-bond character due to resonance, which prevents rotation of peptide bond

Phi and Psi angles

rotation is allowed around bonds linking amide and carbonyl to alpha carbon

- phi: amide

- psi: carbonyl

What do psi and phi angles minimize?

minimize steric clashing = most stable when functional groups are trans

Secondary sequence

Periodic regular structures, folded

Alpha helix

- 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)

- glu

- ala

- leu

- met

- gln

- lys

- arg

beta strands/sheets

- 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)

- val

- ile

- try

- cys

- trp

- phe

- thr

typically too bulky for alpha helix

beta turns

- 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?

- 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

folding of secondary structures into defined protein motifs and domains

How do domains function?

can function independently

ligands function

can bring distinct regions together

disulfide bonds in 3 and 4 structure

can stabilize

Quartenary structure

Assembly of distinct chains into multi-subunit structures (multiple polypeptide chains)

how do different subunits arise

1. different genes coded for diff subunits

2. post-translational modifications

how are different polypeptide chains held together in 4 structure?

- mainly non-covalent interactions

- some can be held together by disulfide bonds

what is the driving force in protein folding?

hydrophobic effect, hb

what is the role of chaperone proteins in protein folding?

bind to exposed/non-folded proteins to prevent aggregatioin

2 cystEINES form disulfide bond which is called?

cysTINE

Preparation of crude extract steps

1. cell lysis

2. centrifuge

how to cell lysis (5)

- grinding

- sonication (high frequency)

- vortexing with glass beads

- osmotic pressure (water flow into cell to break)

- chemical basis (detergents)

considerations of cell lysis

- 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

- proteins separated by size

- column: small molecules enter beads, larger ones elute out

- calibration is required (with known MW)

elution volume

fraction where protein comes off column

fractionation range

range of MW where you can separate 1 protein from another

Vo

- void volume

- anything larger than column's fractional range will go straight through

Ve

- elution volume of a molecule (where molecule comes off column)

Vt

total volume of column (lowest range of fractionation where smallest molecule spend the most time)

area under curve of elution profile

total amount of protein coming off column

how to choose column

between vo and vt

protein absorbance

- 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

molar extinction coefficient depends on number of aromatic aa present (mainly trp and tyr)

ion-exchange chromatography

- separates based on net charge

- cation exchange: elutes catioins

common cation exchanger

carboxymethyl

common anion exchanger

DEAE

how to elute proteins from ion exchange column

- increase salt: compete for binding

- change pH: disrupt HB

affinity chromatography

proteins bind based on affinity for certain molecules

resin of affinity chromatography

resin contains covalently bound. molecules/ligands that recognizes certain proteins in mixture, interacts via non-covalent interactions

how is protein eluted from affinity column

passed through solution of free molecules that compete for binding

example of affinity chrom

his tags + nickel NTA

- addition of 6 his residues at start or end of protein

- use free imidazole can be used to elute

dialysis

- can be used to remove small molecules

- can help get rid of high salt or other molecules used for purification

RP-HPLC

- achieves high resolution of peaks

- RP is based on hydrophobicity

- more hydrophobic = longer in column

SDS-page

- visualization technique

- SDS denatures protein --> coats molecule with negative charge so moves towards end

- smaller molecules move faster

Edman degradation

- can sequence aa

- only up to 100 aa

steps of edman degradation

1. n-terminus labeled with PITC

2. low pH used to break peptide bond (creates cyclic PTH derivative of aa)

3. derivative is separated and analyzed

4. repeat

limitations of edman degradation

- only up to 100 aa

- post-translational modifications may block n-terminus

- proteases may cleave protein to get smaller fragments -- too time consuming

type 5 membrane protein

identifying: western/immuno blotting

1. proteins separated by SDS

2. primary antibody (specific for protein of interest) added to recognize linear sequence

3. secondary antibody (specific for 1st antibody) binds to 1st antibody (produces chemi-luminescent product or fluorescent tag)

4. reveals whether or not protein is present, potential size

identifying: mass spec

1. peptides bombarded by high energy electron beam/laser

2. attracted to charged plate detector, analyzed for time of flight

3. based on charge to mass ratio, can identify protein

4. highly charged or smaller molecules will travel faster

Tryptophan fluorescence

- presence of indole ring allows Trp to fluoresce when excited around 280 nm

- sensitive to polarity

Trp red shifted

polar environment - 350 nm