Biochemistry Midterm Prep

4 major classes of biological molecules

amino acids, carbohydrates, nucleotides, lipids

3 major polymers

protein, polysaccharides, nucleic acids

central dogma of molecular biology

how genetic info is stored, retrieved in living cells and transmitted to offspring

polymers of amino acids

DNA

polymers of carbohydrates

polysaccharides

polymers of nucleotides

nucleic acids

molecular range of proteins

5 kilodaltons to 1000+ kDa

function of proteins

catalysts, structural, transport, storage, signalling etc.

synthesizing components of proteins

20 amino acids

enthalpy (H)

heat content of system

entropy (S)

system’s disorder/randomness

Gibbs free energy

free energy of system based on H and S

∆G < 0

reaction spontaneous

∆G and what its dependent on

= G products - G reactants

depends on

• chemical nature of molecule

• environmental conditions (temperature, pressure and concentration)

determining factors of shape and function of protein

genetic sequence

difference between amino acids

side chains

2 forms of amino acids

L-forms and D-forms

Form of amino acids incorporated into proteins

L-forms

Condensation reaction and amino acids

condensation reaction form peptide bonds forming a link between 2 amino acids

peptide bond

a covalent bond forms between alpha-amino group of one amino acid and the alpha-carboxyl group of another

N-terminal

1st amino acid of the polypeptide chain, which still has amino group because it uses carboxyl group to form bond with 2nd residue

C-terminal

last amino acid of the polypeptide chain consisting of a carboxyl group

frequent usage of certain amino groups

common because they may serve more common and important roles

Hydrophobic amino acids

• Alanine (Ala, A)

• Valine (Val, V)

• Phenylalanine (Phe, F)

• Tryptophan (Trp, W)

• Leucine (Leu, L)

• Isoleucine (Ile, I)

• Methionine (Met, M)

• Proline (Pro, P)

  • Glycine (Gly, G)

Polar amino acids

• Serine (Ser, S)

• Threonine (Thr, T)

• Tyrosine (Tyr, Y)

• Cysteine (Cys, C)

• Asparagine (Asn, N)

• Glutamine (Glu, Q)

  • Histidine (His, H)

Charged amino acids

• Aspartarte (Asp, D)

• Glutamate (Glu, E)

• Lysine (Lys, K)

• Arginine (Arg, R)

simplest amino acid

glycine

Disulfide bond

formed when cysteine’s thiol group undergoes oxidation with another thiol group, such as another cysteine side chain, irreversible bond

normal pH in body

7.0

Dipeptide

a peptide chain composed of 2 amino acids

Tripeptide

a peptide chain composed of 3 amino acids

oligopeptide

a peptide chain composed of approx. 3-20 amino acids

polypeptide

a peptide chain composed of many amino acids

protein

a molecule composed of one or more polypeptide chains

primary structure of protein

the sequence of amino acid residues

secondary structure of protein

the spatial arrangement of the polypeptide backbone

tertiary structure of protein

the 3D structure of an entire polypeptide, including all its side chain

quaternary structure of protein

the spatial arrangement of polypeptide chains in a protein with multiple subunits; if protein only has one polypeptide chain then it has no quarternary structure

Stabilizing factor of secondary structure of proteins

stabilized by hydrogen-bonds between backbone N-H and C=O groups

two major types of secondary structure

alpha-helix and beta-sheet (consists of beta-strands)

protein that doesn’t have “regular” secondary structure

proline, doesn’t have amide H to participate in stabilizing the bonds

hydrogen bond formation in secondary structure alpha-helix

carbonyl oxygen of each residue forms a h-bond with backbone NH group four residues ahead

Aº rise of the helix

helix rises 5.4Aº along its axis per turn

“Kink” in proline structure

if residue 9 is proline it will not form H-bond with residue 5, because no amide H, but if residue 5 is proline, then it will form H-bond with residue 9, depends of location of proline in sequence

Reason why glycine is not frequently used

tends to be in a more flexible location, thus when in an secondary structure, it usually destabilizes the secondary structure

number of residues per turn

≈3.6 residues pe’’r turn

H-bonds in the secondary structure of the beta-sheet

formed between neighbouring strands

proteins that can form the beta-sheet secondary structure

any protein because all of them have backbone atoms

antiparallell beta-sheet

parallel beta-sheet

determining factors of secondary structures interaction

side chain because side chains point out of alpha-helix, if another helix comes close they can bond to their side chains packing them together

stabilizing factor in tertiary structure

stabilized by interactions between side chains and backbone atoms and interactions between residues, however the interaction between residues are distant in sequence

reason of difficulty in degrading proteins

strong bonding like covalent, ionic, hydrogen, and Van Der Waals

stronger bond type in aqueous solution

covalent bond stronger than ionic bond

5 representations of protein structures

1) backbone model

2) ribbon model

3) wire model

4) space-filling model

5) electrostatic potential model

backbone model of protein structure

shows only backbone structure showing N-terminus

ribbon model of protein structure

used to see alpha-helices and beta-sheets

wire model of protein structure

shows bonds and atoms in detail but doesn't show how much space they take up

space-filling model of protein structure

show the shape and form of macromolecules

electrostatic potential map of protein structure

shows charge on surface of protein

• red = (-) charge

• blue = (+) charge

Stabilizing factor in quaternary structure

noncovalent bonds

• h-bonds (backbone-backbone, backbone-side chain, side chain-side chain,_

• ionic bonds

• van der waals

covalent bonds

• disulphide bonds

• other bonds

example of quaternary structure

hemoglobin

• a hetero-tetramer with 2 alpha subunits and 2 beta subunits

hydrophobic effect on protein folding

usually core of protein is mainly hydrophobic and outside is hydrophilic (polar/charged)

change in water molecules when hydrophobic group is present

water molecules can rotate in many directions without losing h-bonding partners, however when exposed to hydrophobic group, water molecules are more restricted in its rotation

• each hydrophobic side chain reduces the freedom of two water molecules

correlation between hydrophobic side chains and entropy

side chains decrease entropy as they restrict rotation of water molecules

protein folding conformation

initially is folded randomly then progresses to a more ordered conformation

3 fates of misfolded protein

1) aggregation

2) refolding

3) degradation

chaperone proteins

help proteins fold by binding to hydrophobic groups to stabilize proteins

denaturation

unfolding of protein thus destroying structure of protein

chemical denaturation

adding chaotropic agents in high concentrations which increases the solubility of non polar substances in water leading to protein unfolding

thermal denaturation

applying heat leads to the proteins having so much energy that the bonds in these proteins cannot be kept together causing them to denature

protein domains

distinct region of a protein that can often often fold independently and provide structure and/or function by interacting with one another

• related domains often found in different proteins

protein family

proteins related by evolution and have similar primary sequences, functions, and domains

conserved residues

residues that are critical for function do not change on an evolutionary timescale

Chromatography

a process for separating components of a mixture (proteins)

gel filtration

type of chromatography that separates proteins by size

• consists of a column with gel beads that have gel matrixes inside them that allow small molecules to enter internal spaces of gel beads slowing them down (reducing elution speed)

• larger molecules come out of the column early

  • elution speed also affected by protein shape

ion exchange

type of chromatography that separates proteins based on charge

• consists of a column with charged mixture, if (+) then (-) proteins will bind to it and (+) will come out of column

  • to then get the protein increase salt concentration

isoelectric point (pl) and charge on protein

if pH= pl → charge on protein is 0

if pH<pl → charge of protein is (+)

if pH>pl → charge of protein is (-)

Affinity Chromatography

separation based on molecular interactions

• relies on interaction between a protein and a ligand

• if ligand is attached to column matrix, certain proteins will bind to it, others won’t

  • flow of buffer will wash away unbound proteins, leaving only the specifically bound proteins

SDS-PAGE

SDS Polyacrylamide Gel Electrophoresis is an analytical method used to analyze what proteins are in the mixture

• consists of sodium dodecyl sulphate (SDS), an ionic detergent used to denature proteins and gives them (-) charges upon binding

  • charged molecules migrate in an electric field in which the gel acts as a molecular sieve and allows smaller molecules to go faster, thus separates on the basis of polypeptide chain size

mass spectrometry

provides info on identity (sequence), abundance, and various modification

• an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one or more molecules present in a sample

• sample gets ionized, then separated by charge and mass, separated ions are then measured and results are displayed

X-ray Crystallography

type of structural analysis which determines 3D structure of proteins

• protein must be crystallized first and then x-ray is taken displaying X-ray diffraction patterns

NMR spectroscopy

Nuclear Magnetic Resonance spectroscopy determines the 3D structure of relatively small proteins, in this crystallization of the protein is not needed

• ensemble of structures is obtained

Post translation modifications discussed in class?

phosphorylation, ubiquitination, acetylation, sumolyation → alters stability or signalling

glycosylation → affects protein folding, secretion, solubility, binding to other biomolecules

myristoylation, farnesylation → alters location

Amino acids that can be phosphorylated

serine, threonine, tyrosine

protein-ligand binding

many proteins contain sits to which ligands specifically bind and form a “complex” with the protein

• this binding occurs by multiple weak and few strong forces

  • ligand binding is reversible depending on affinity and concentration of ligands

Dissociation constant

Kd (dissociation constant) is a measure if strength or affinity of an interaction

Kd = ([P][L])/[PL]

P = Molar concentration of free protein

L = Molar concentration of free ligand

PL = Molar concentration of protein-ligand complex

units = moles/L (M)

Kd and strength of binding

smaller Kd = tight binding = high affinity

% of ligand in binding form

f = [PL]/[P] + [PL]

myoglobin

oxygen-binding protein consisting of 8 alpha-helixes plus one heme prosthetic group, which facilitates oxygen diffusion through tissues

heme group

its a certain group that only after it binds to myoglobin can it allow myoglobin to bind to oxygen, this heme group

Atom in heme group that binds to oxygen

iron form coordination bond with oxygen in myoglobin

equation that measures the fraction of binding sites that are occupied in myoglobin

YO2 = pO2/(K + pO2)

Kd = ([Mb][O2])/[MbO2]

K = pO2

YO2 = 0.5 → half of the binding sites are occupied

K = p50

The O2 pressure at which Mb is 50% saturated

pO2 > K

means almost 100% will be in binding form (myoglobin + oxygen)

At a pO2 of 10 torr, what fraction of myoglobin molecules have an O2 bound?

0.78

hemoglobin

has 4 subunits, two alpha subunits and two beta subunits, plus heme groups, each subunit can transport 1 O2 molecule, thus 4 all together

percentage of how identical Mb and Hb are

only about 18% in primary sequence

explain the graph and its discrepancies

In the beginning Hb has a low affinity to O compared to Mb, thus dotted-line myoglobin + O2 in a hyperbola shape, and solid line hemoglobin + O2 in a sigmoid