Biochem Midterm 1

Key organelles for plant and animal cell

• Both:

  • Nucleus: contains chromosomal DNA

  • Mitochondrion: energy generation

• Plant:

  • Chloroplast: photosynthesis

• Animal:

  • Lysosome: protein degradation

Key organelles and function for malaria parasite

Microneme and rhoptry contain proteins which are essential for host cell invasion by malaria parasite

Relevance of lipids for cells

Lipids form membranes that surround the cell and separate organelles

Advantages of compartmentation

• Protect rest of cell from harmful events inside organelle (lysosome enzymes)

• Create specialized membrane bound machineries inside cells (photosynthesis)

Composition of cells

70% water, 15% proteins (lots of variety), 1,5% DNA/RNA

Major biopolymers & monomer makeup

Proteins (amino acids), nucleic acids (nucleotides), polysaccharides (monosaccharides)

Building block of polysaccharides, continuous linkages name, how to make glycogen

• Building block is α1,4 linked D glucose units

• α-amylose is continuous connections

• Adding α1,6 branches give glycogen

Free energy equation, G0, when is spontaneous

• delta G = delta H - T delta S

• delta Go = -RT ln(Keq)

• G-: spontaneous, exergonic

• G+: non spontaneous, endergonic

Entropy and Enthalpy

• Entropy moves towards disorder

• Enthalpy moves towards order

Rank non covalent interactions

Charge charge > dipole dipole > dipole induced dipole > dispersion

Important H2O bond length

H-bond distance w/ O-H is 1.77 A vs sum of van der waals = 2.6 A

Hydration shell definition

Dipoles of water interact favorably with charged ions in solution

Hydrophobic effect/interactions & amphiphilic/amphiphatic definition

• Hydrophobic interactions: cause non polar groups to come together in H2O, decreasing exposed SA to H2O, increasing H2Os entropy/movement

• Amphiphilic: both polar and non polar segments

Hydrocarbons and water to non polar solvents reaction

• Enthalpy is 0, so entropy drives process

• Entropy is smaller for hydrocarbon in H2O than when by other hydrocarbons

Micelles and Bilayers

• Miscelle: all hydrophobic groups are sequestered from water; sphere

• Bilayer: 2 layers of molecules forming a sheet, hydrophobic hidden between

Find moles of H2O and H3O+/OH- and Kw for 1L H2O

• 55.5 moles H2O in 1L

• [H3O+/OH-] = 10^-7

• Kw = 10^-14

Equation for pH and HH

• pH = -log[H+]

• pH = pKa + log([B]/[A])

How does Influenza virus use pH

Virus enters endosome where pH is low → haemagglutinin conformational change → fusion & infection

Typical structure of amino acid + zwitterionic

• Alpha carbon w/: H, R group (side chain), amino group (NH2), carboxyl group (COOH)

  • NH2 = amino terminus, start ;COOH = c terminus, end

• Zwitterionic: Amino group is protonated (NH3+), carboxyl group is deprotonated (COO-)

Which amino acids are non polar, polar neutral, and polar charged

Non polar: Grandma Always Visits London In May For Winston’s Party

Polar neutral: Santas Team Crafts New Quilts Yearly

Polar charged: Dragons Eat Knights Riding Horses

pKas for amino acids needed

DEHCKYR

3.9, 4.2, 6.0, 8.3, 10.0, 10.1, 12.5

Creation of disulfide bridges

2 cysteine residues get oxidized and thiol groups (SH) form covalent bond with each other

2 important amino acid side chain modifications

Phosphoserine: adds phosphate group (PO3 2-) to O side chain

4-Hydroxyproline: adds hydroxyl group (OH) to 4 carbon on ring

Kinases can add phosphoryl groups onto which amino acid side chains

Serine, Tyrosine, Threonine

Protein glycosylation definition, function, types

• When sugars are added to specific residues of a protein

• Forms extracellular matrix (ECM): mixture between cells that positions them properly and is crucial for wound healing and developmental biology

• O-glycosylation: Added to side chain O of Ser or Thr

• N-glycosylation: Added to side chain N of Asn

How is peptide bond made and pKas of N,C terminus

• Condensation; peptide bond between carboxyl and amino terminus via ribosome; O from carboxyl and 2 Hs from amino group leave

• N=8, C=4

Primary, secondary, tertiary, quaternary structures

• Primary: order of amino acids (Lys-Cys-Ala-His)

• Secondary: local spatial arrangements (helix)

• Tertiary: complete protein chain

• Quaternary: interactions between multiple chains

Two main chain torsional angles

• Phi Φ: bond angle between N-C alpha

• Psi Ψ: bond angle between C alpha-C

Ramachandran plot details

• Shows allowed torsional angles for 18 amino acids

• Gly has no side chain so more allowed angles

• Pro has more restricted plot

α-helix angles, residues per turn, height rise per residue, H bonding

• Phi = -57, psi = -47

• 3.6 residues per turn (100 right handed rotation per residue, 360/100=3.6)

• 1.5 angstrom rise per residue

• H bond between C=O of n residue and NH of residue n+4

  • patterns of 3/4 hydrophobic/philic to match 3/6 residues per turn

β-strands types, handed type, sheet handed type

• Parallel: backbones in run in same direction Anti-parallel: backbones run opposite

  • alternating hydrophobic and hydrophilic since residues point in opp directions

• Strands have right hand twist

• Sheet (many strands) has left hand twist

Keratin and coiled coil α-helices structure and residue structure

• 2 coiled α-helix

• a-b-c-d-e-f-g a,d are non polar residues

Collagen structure, handed type, and common residue make-up

• 2 α1 and 1 α2 chains (not α helix) with a right hand twist

• Often Gly-Pro-Hyp

3 secondary structures and 3 domains

• Secondary structures: βαβ-motif, αα-motif, βββ-motif

• Domains (combination of secondary structure elements): All-α, All-β, α/β

  • membrane proteins are either All-α or All-β

4 factors stabilizing native state of proteins

• H bonding: with water in unfolded state

• Van der waals: with water in unfolded state

• Conformational entropy: greater in unfolded state

• Hydrophobic interactions: non polar groups are sequestered in water to inc entropy

What amino acid is helix breaker and why

Proline because its NH can’t make an H bond due to covalent bond

Protein purification procedures for: solubility, size, charge, binding specificity

• Solubility: add salt and centrifuge, hydrophobic precipitate out first

• Size:

  • Selective Dialysis: small proteins diffuse out dialysis bag

  • Gel filtration chromatography: large come out first, small last

• Charge: Ion exchange chromatography; pass thru ion matrix

  • weak anion exchanger: is + and binds -

  • weak cation exchanger: is - and binds +

• Binding: Affinity chromatography; separates based on binding to molecule of interest

Isoelectric point (pI) definition and relation with pH

• pH at which net charge is 0

• If pI > pH, positively charged

• If pI < pH, negatively charged

3 methods to determine protein concentration

• UW spectroscopy: determine absorption of side chains at 280nm for Trp, Tyr (Phe absorbs 250nm)

• Coomassie staining: blue binds proteins and shifts absorbance to 595nm

• ELISA: use antibody w/ enzyme to detect specific protein

Methods for protein characterization

• SDS Page: denatures & binds protein → run in electrophoresis (large moves slower)

• Mass Spec:

  • ESI: determines mass-charge (m/z) ratio

    • shoulders due to heavier isotopes

  • Tandem MS: separate into fragments and fun MS again

Amino acid sequence determination methods

• Protease: break peptide bond based on adjacent amino acid (don’t cleave if Pro)

  • Trypsin cleaves Arg, Lys

  • Chymotrypsin cleaves Phe, Trp, Tyr, Leu

Protein sequence analysis method

Sequence alignment: align sequences and determine how many amino acids match

• gaps/indels to maximize identities

What is point group symmetry and what are the 3 types

• Point group symmetry: all symmetry axes intersect at one point in center or particle

  • Cyclic (C): rotational symmetry about n-fold axis, Cn = #

  • Dihedral (D): rotations and reflections about multiple axes, D2n = #

  • Polyhedral (T,O,I):

    • Tetrahedral: 12 same subunits, 2/3 fold symmetric axis

    • Octahedral: 24 same subunits, 2/3/4 fold symmetric axis

    • Icosahedral: 60 same subunits, 2/3/5 fold symmetric axis

Definition and difference for helical symmetry vs point group with example

• Subunits make a segment which is non-covalently linked to other segments, includes rotation and translation, has more fluidity

• TMV virus has helical symmetry

Actin function

• Actin polymerization leads to movement

• G-actin: 4 domain protein that is assembled into microfilaments

• F-actin: microfilaments, 2 right handed strands of g-actin

AB5 toxin makeup and examples

• B-pentamer + A-subunit; B binds and A modifies protein

Function of PDI

• Helps proper formation of disulfide bonds

• Wrongly connected: will fix bond, starts and ends reduced

• Not yet connected: starts oxidized and ends reduced

GroEl/ES chaperone structure and folding process

• GroES cap, GroEL cis and trans ring

• GroES cap binds GroEL → cis ring made → protein refolds → 2nd improper protein binds trans ring → GroES, ADP, proper protein released

Folding disease cause and example

• Caused by protein misfolding, forming fibrous aggregates called amyloids

• Alzheimer’s: β-strands crosslink with amyloid fibers making them hard to remove

Myoglobin purpose, structure, oxygen curve shape

• Waits in muscle cells for oxygen to arrive

• 8 helices, heme Fe group attached to 4 Ns, O, His

• Oxygen binding curve is hyperbolic

Hemoglobin purpose, structure, 2 states, O2 binding result

• Transports oxygen through blood

• 2 α chains and 2 β chains

• Deoxy = T state Oxy = R state

• O2 binds → Heme becomes planar → trigger for T to R state

Oxygen binding curve for hemoglobin, relation to Hill eq, Bohr effect

• Hemoglobin: sigmoidal (S) due to cooperativity

• Hill equation describes S shape, larger Hill coeff = more steep

• Bohr: as pH inc → His more protonated → deoxy more favorable → less O2 binding

Allosteric effect definition, symmetric model, sequential model

• Allosteric effect: binding of one ligand affect binding of other ligands at other sites

• Symmetric model: subunit can either be R or T

• Sequential model: ligand binding induces conformational change for other subunits, symmetry not needed

BPG function, net effect, effect on oxygen curve

• Binds deoxy Hb and decreases affinity for O2

• Delivery of O2 is very efficient

• Shifts O2 binding curve right

Hemocyanin purpose, symmetry, O2 binding structure

• Oxygen binding protein for mollusks/arthropods

• Hexamer with D3 symmetry

• O2 bound by 2 Cu liganded by 6 His

Sickle cell anemia cause, physical effect, connection with malaria

• Mutation of Glu6 to Val6 leading to different interaction

• Very rigid RBCs

• HbA:HbS protects against malaria, HbS homozygote dies, HbA homozygote gets malaria