Chapter 1

-Fe involved in the transport of oxygen and in redox enzymes

-Cu, Z, Se, Co are important at the active site of certain enzymes

Amino acids = proteins

Monosaccharides = starch, glycogen, chitin, cellulose

Nucleotides = DNA, RNA

Lipids = membranes

Important Reactions

Alcohol + acid -> ester

Thiol + acid -> thioester

Alcohol + aldehyde -> hemiacetal

Alcohol + ketone -> hemiketal

Amine + acid -> amide

Thermodynamics

-used to determine whether a physical process is possible

Enthalpy: H, measure of heat evolved during a reaction

Entropy: S, measure of disorder

Free Energy: G, measure of the tendency for a process to occur

Bioenergetics: study of energy transformation in living organisms

1st law-> energy is conserved

2nd law -> spontaneous processes are characterized by the conversion of order to disorder

-entropy increases as the temperature rises (k is dependent on temperature)

Gibbs free energy

G=H-TS

G=-RTlnK

ATP + H2O = ADP + Pi exothermic

ATP + H2O = AMP + Pi exothermic

-exergonic because of resonance stabilization, electrostatic repulsion, smaller solvation energy

Chapter 2: Water and Aqueous Solutions

-oxygen has a partial charge of -0.82 and hydrogen has a partial charge of +0.41

-causes a permanent dipole moment of 1.85 Debye units

-Hvap is the energy needed to break intermolecular interactions, increases as MW increases

H bonds -> 1.8A long, strongest when OH-O is co-linear

Co-operativity: probability that a second H bond will form after the first is increased

-H2O is a good solvent because it is highly polar and has H bonding capability

Coulomb’s law: describes the electrostatic forces between charged molecules

Colligative properties of H2O -> properties of solutions that depend on the number of molecules in a

given amount of solvent, not on the identity of the molecules

-lowering of VP, lowering of freezing point, elevation of boiling point, osmotic pressure

-in dilute solutions the effect is directly proportional to the number of solute particles per unit volume

Osmotic pressure: pressure generated by the mass flow of water to that side of a membrane-bonded

structure that contains the higher conc of solute molecules

Osmosis: water movement across a semipermeable membrane driven by differences in osmotic

pressure

1. Isotonic – equal osmolarity, no net flow

2. Hypertonic – cell is in a solution with a higher molarity, cell shrinks as water flows out

3. Hypotonic – cell is in a solution with a lower molarity, cell expands as water flows in (lysis)

Erythropoietin -> glycoprotein hormone that stimulates the production of oxygen carrying red blood

cells, it is a cytokine for erythrocyte production in the bone marrow, it’s made by fibroblasts in the

kidney; failing kidneys no longer produce sufficient EPO

Buffers: solution containing substantial conc of a weak acid and its conjugate base which resists changes

in pH upon addition of acid or base

Buffer capacity: number of mols of OH- that must be added to 1L of a solution to increase it’s pH by 1

unit

Buffer ratio = Cb/Ca

-a buffer is most efficient when the buffer ratio is 1 (Cb=Ca)

Chapter 3: Nucleic Acids and Genetic Info

Avery, Macleod and McCarty -> reported that DNA alone can transform nonpathogenic R-type

pneumococci into the virulent S form

Erwin Chargraff’s Rule -> A=T; G=C, the base composition of a given organism is independent of the

source of tissue

Hershey-Chase experiment -> only the nucleic acid component of bacteriophages enters the bacterial

host during phage infection (protein labeled with S, nucleic acid with P)

Watson & Crick -> B-DNA, right-handed twist, 20 A in diameter, sugar phosphate backbone, 10bp/turn

(34A), planes of bases are perpendicular to helix axis

Meselson & Stahl -> semiconservative nature of DNA replication by Cs Cl gradient (1st replication was a

hybrid, the 2nd was half hybrid, half new)

Stop codons = UGA, UAG, UAA

-DNA double helix is held together by stacking interactions between bases

Tm (melting temperature) of DNA is dependent on: base composition, pH, ionic strength

-denaturation of DNA does not change the shape of absorbance curve but it does increase its intensity

due to increased exposure

-DNA annealing rate is dependent on length of dsDNA

-ribosomes synthesize polypeptides, 2/3 RNA and 1/3 protein

-RNA is more susceptible to base-catalyzed hydrolysis since its bases are more exposed

Site-directed mutagenesis -> a primer is synthesized containing mismatches corresponding to the

desired mutations and hybridized to the WT to create an altered gene

Molecular Cloning

Type I&II RE: have both endonuclease and methylase activity

Type I cleaves at random site 1000bp from recognition sequence

Type III cleave 24-26bp from recognition sequence

Type II do not have methylase activity, recognize 4-8bp sequences that are palindromic

Isoschizomers: enzymes that have the same recognition sequence but do not cleave at the same site

Isocaudomers: enzymes that produce identical sticky ends

Maxam-Gilbert method -> uses chemicals to break bases for sequencing

Human genome – 90% done, half is repeating sequences, 28% is transcribed into RNA, 1% codes for

proteins, 30 000 ORFs

Reporter genes -> used to monitor transcription activity (green fluorescent protein)

Xeroderma pigmentosum – genetic disease results in a lack of nucleotide excision repair, extremely light

sensitive, easily develop cancer

PCR -> invented by Kary Mullis, may be used for DNA, cloning, mutagenesis, sequencing, methylation,

genotyping, etc.

Chapter 4: Amino Acids

Isoelectric pH: pH at which a molecule has a net charge of zero; can be useful when trying to purify a

protein

Source of amino acids -> hydrolysis of proteins, fermentation or synthetically made -> hydrochloride

salts, sodium salts, isoelectric form, esters, amides

-AA are historically related to glyceraldehyde (sugars)

Sinistrus = L (counterclockwise)

Rectus = D (clockwise)

-chiral AA are all L except for Cys which is D form and Gly which is not chiral

Hydrophobic AA -> alanine (a, ala), leucine (L, leu), valine (v, val), isoleucine (I, ile), phenylalanine (F,

phe), tryptophan (w, trp), methionine (m, met), proline (p, pro)

AA with uncharged polar side chains -> Serine (s, ser), threonine (t, thr), tyrosine (tyr, y), cysteine (c,

cys), asparagine (asn, n), glutamine (q, gln)

AA with charge polar side chains -> aspartic acid (d, asp), glutamic acid (glu, e), lysine (K, lys), arginine

(arg, r), histidine (h, his)

Hydropathy: the relative hydrophobic tendency of AA

Chapter 5: Proteins – Primary Structure

Protein Purification

Methods of solubilization -> osmotic lysis, homogenization, sonicator, French press, differential

centrifugation

Protein stability dependent on -> pH, temp, proteases, adsorption to surfaces, storage

Protein Quantification

Immunochemical assays – generation of antibodies extracted from blood serum; involves the fusion of a

cell producing a specific antibody with a myeloma cell

Separation of AA mixtures

Protein Characteristic Purification Procedure

Solubility Salting out

Ionic charge Ion exchange chromatography, electrophoresis, isoelectric focusing

Polarity Hydrophobic interaction chromatography

Size Gel filtration chromatography, SDS-PAGE

Binding specificity Affinity chromatography

-Phe (260nm), Tyr (276nm) and Trp (280m) are visible in UV light

Beer-Lambert Law:

-peptide bond absorbs strongly in the far UV with a max at 190nm

-must either remove oxygen or take absorbance at 205nm

-absorbance is proportional to length of the cuvette

-the more AA you have, the more absorbance at 570nm except for Pro (440nm max)

-in Ninhydrin AA turn purple, except for Pro which turns yellow (doesn’t have a primary amino group)

-dansyl chloride reacts with N-terminus and in highly acidic conditions peptide bonds are broken

liberating the dansylating N-terminal residue which can then be separated by chromatography and

identified by its fluorescence

Bradford assay -> the binding of the dye Coomassie brilliant blue to proteins in acidic solution causes

the dye’s absorption maximum to shift from 465nm to 595nm; hence the absorbance at 595nm provides

a direct measure of the amount of protein present (highly sensitive, detects as little 1ug of protein per

mL)

Radioimmunoassay (RIA) -> the protein is indirectly detected by determining the degree to which it

competes with a radioactively labeled standard for binding to the antibody – 1. Sample is added to the

well, time is allowed for antigens to bind to walls of the well 2. Radioactive antibodies, specific to the

antigen are added to the well 3. If the antigens are present the antibodies will bind 4. Washing removes

unbound antibodies 5. Detection of radiation allows you to see if the antigen is present

ELISA (enzyme-linked immunosorbent assay) -> 1. Antibody against the protein of interest is

immobilized on an inert solid like polystyrene 2. The solution to be assayed is applied to the antibody-

coated surface; the antibody binds the protein of interest and other proteins are washed away 3. The

protein antibody-complex is reacted with a second protein-specific antibody to which an enzyme is

attached 4. Binding of the second antibody-enzyme complex is measured by assaying the activity of the

enzyme; the amount of substrate converted to product indicates the amount of protein present

Isolation of Biological Molecules

1. Solubility

Salting in –the solubility of a protein at low ion concentrations increases as salt is added; the additional

ions shield the protein’s multiple ionic charges, thereby weakening the attractive forces between

individual proteins

Salting out – as more sulphate salts are added, the solubility of the protein decreases; result of

competition between the added salt ions and the other dissolved solutes for molecules of solvent; at

very high salt concentrations, so many of the added ions are solvated that there is significantly less bulk

solvent available to dissolve other substances, including proteins

-ammonium sulphate is often used due to its high ionic strength

-organic solvents like acetone and ethanol are used to precipitate proteins

-the solubility of a protein is at its lowest at its pI

2. Ion Exchange Chromatography

-charged molecules bid to oppositely charged groups that are chemically linked to matrix such as

agarose or cellulose

-anions bind to cationic groups on anion exchangers (diethylaminoethyl) and cations bind to anionic

groups on cation exchangers (carboxymethyl)

-those that do not bind are eluted first then those that bind are eluted using increasing salt

concentrations to increase ionic strength

3. Gel Filtration Chromatography

-larger molecules cannot enter pores and come out first leaving the smaller molecules to come out

second

-separates based on MW and size

4. Affinity Chromatography

-a ligand that specifically binds to the protein of interest is covalently attached to an inert matrix

-when an impure protein solution is passed through its chromatographic material, the desired protein

binds to the immobilized ligand whereas other substances are washed through the column with the

buffer

-the desired protein can be recovered in a highly purified form by changing the elution conditions to

release the protein from the matrix

5. Paper Chromatography

-partition an AA between a polar stationary phase (support, paper, silica gel) and a mobile phase

(solvent phase)

-more polar substances will be retained more favourably by the polar support and migrate at different

rates

Kp = conc in mobile phase/ conc in stationary phase

1. Apply sample

2. Put in container with solvent, by capillary action the solvent migrates up

3. After solvent reaches the top of plate, plate is removed and analyzed by visualization with

ninhydrin

Rf = distance traveled by substance/distance traveled by solvent

6. Metal-Chelate Affinity Chromatography

- a metal ion like Zn2+ or Ni2+ is attached to the chromatographic matrix so that proteins bearing metal-

chelating groups can be retained

-commonly uses Zn2+, Ni2+, Cu 2+ to form stable complexes with histidines, tryptophan and cysteine

residues within proteins

7. Hydrophobic Interaction Chromatography

-stationary phase only lightly substituted with hydrophobic groups like phenyl groups

-can retain the native structure of proteins

-at high concentrations, nonpolar groups on the surface of proteins interact with the hydrophobic

groups; both types are excluded by the polar solvent

-the eluent is in an aqueous buffer with decreasing salt concentrations, increasing concentrations of

detergent (disrupts hydrophobic interactions) or changes in pH

8. High Performance Liquid Chromatography

-performed under high pressure

-better resolution, speed, sensitivity and automation

9. Electrophoresis

-migration of ions in an electric field

-size separation

-depends on charge, shape, voltage

10. Discontinuous pH Method

-includes a stacking gel with a pH 2 units lower than running gel

-upper reservoir contains glycine inn buffer in its zwitterion form creating high resistance

-much better resolution (small pores)

11. Polyacrylamide Gel Electrophoresis

-SDS unfolds proteins and gives a constant mass/charge ratio

-can estimate within 5% of the MW of your protein

12. Isoelectric Gel Electrophoresis

-separates proteins based on their net charge

-urea is used to denature proteins (unfolded but retains charge)

-protein migrates to its pI point

SDS-PAGE -> sodium dodecyl sulphate (SDS) is used to denature proteins by interfering with

hydrophobic interactions that normally stabilize proteins, the SDS negative charge masks other charges

so proteins are separated purely by mass; to test if subunits are linked by disulfide bonds, samples can

be prepared using SDS-PAGE in the presence and absence of a reducing agent like (2-mercaptoethanol)

which breaks these bonds

2D gel electrophoresis – combines isoelectric focusing and SDS page to compare proteins based on both

MW and charge

Amino acid composition -> 6M HCl, high temp to hydrolyze peptide bonds (Trp is destroyed, Cys, Ser, Thr

are partially destroyed, Asn and Gln are de-aminated)

-2M KOH, high temp destroys most AA except Trp

Amino acid analyzer – separates AA by ion exchange chromatography using and HPLC

-AA are fluorescently labeled by treatment with dansyl chloride or Edman’s reagent

Determination of a Purified Protein

1. Reduce and alkylate (prevents reformation) of disulfide bonds

2. Fragment with endopeptidase or CNBr and separate each fragment

3. Characterize each fragment by AA analysis or sequencing

4. Overlap fragments

5. Deduce positions of disulfide bonds

1. Chemical cleavage of disulfide bond -> reversible reduction using 2-mercaptoethanol or

dithiothreitol; irreversible reduction using iodoacetate or performic acid

2. Peptide Cleavage

a. Enzymatic hydrolysis by proteases

Endopeptidase: any proteolytic enzyme that cleaves an internal protein peptide bond

Exopeptidase: any proteolytic enzymes that cleaves the terminal bond in a peptide chain

Carboxypeptidase – C-terminal residue

Aminopeptidase – N-terminal residue

b. Chemical cleavage of peptide bond -> CNBr cleaves at the C-terminus of Met

Enzyme Specificity Comments

Trypsin R(n-1)=positive charged residues: Arg, Lys

Rn=/ Pro Highly specific

Chymotrypsin R(n-1)=bulky hydrophobic residues: Phe, Trp, Tyr

Rn=/ Pro Cleaves more slowly for R(n-1)=Asn, His, Met, Leu

Thermolysin Rn=Ile, Met, Phe, Trp, Tyr, Val

R(n-1)=/Pro Occasionally cleaves at Rn=Ala, Asp, His, Thr; heat stable

Pepsin Rn=Leu, Phe, Trp, Tyr

R(n-1)=/Pro Nonspecific

Endopeptidase R(n-1)=Glu

3. Protein Sequencing

-dansyl chloride reacts with NH3+

-lysine has NH3+ side chain which can be misleading

-Edman’s degradation involves PITC reacting with N-terminus to form PTC which is then treated with

trifluoroacetic acid to cleave the N-termini residue

Mass Spectrometry

-mass/charge ratio in gas phase

Electrospray ionization -> complex mixtures, produces gas-phase ions from solution and can be

integrated with capillary electrophoresis liquid chromatography

-dissolved and forced through a narrow needle held at high voltage, droplets dried by nitrogen gas

MALDI (matrix assisted laser ionization) -> analysis of simple peptide mixtures

-mixed with aromatic matrix compound which can absorb energy from the laser

-dissolved in organic solvent and transferred to target

-organic solvent evaporates ad leaves matrix crystal with embedded analyte

-placed under vacuum ad pulsed with laser and voltage

-converts analyte into gas phase ions that accelerate towards detector

P1 = (M+z)/z

P2= (M+z-1)/(z-1)

Peptide sequencing -> short AA (less than 25) can be sequenced by 2 mass spectrometers coupled in

series

-proteins are cleaved into 2 fragments then bombarded by He in a collision cell and a detector reads out

all of the different fragments

Protein Evolution

1. Invariant residues – key indication that AA are important for function ex. Cytochrome C, histone

H4

2. Conservatively substituted – likely important for function but have some flexibility

3. Hypervariable – change often, aren’t key to function

Chapter 6: Proteins and 3D Structure

-some residues are more frequent than others

-approx 50-9000 AA per polypeptide; the average molecular mass is 110 Da

-MW would be 5500-100 000 Da per polypeptide

-polypeptide chains are joined by disulfide bridges and/or noncovalent forces

Primary Structure -> AA sequence of the polypeptide chain(s)

-peptides are <40 AA while polypeptides are >40

-the majority of polypeptides are 100-1000 residues

Classification of Proteins

Shape -> globular – spherical and soluble

Fibrous – rod-like and insoluble

Function -> enzyme, transport, contractile, structural, defense, regulatory

Prosthetic Group (group attached to protein) -> glycoprotein (carbohydrate), lipoprotein (lipid),

nucleoprotein (nucleic acid), metalloprotein (metal), heme protein (heme)

Secondary Structure -> local conformation of polypeptide chain

-peptide bond has 2 resonance forms and (40%) partial bond character; usually in the trans

configuration

-planar structure restricts the freedom of rotation so there is no rotation around the C-N bond

-limited by the steric interference between adjacent residues

-peptide bond (N-Ca-C) is trigonal with 120 bond angles

-Pro has more of the cis conformation than any other AA, about 10% of the time

-Gly has the greatest conformational freedom

The Alpha Helix

Helical pitch (p) – distance the helix rises along its axis per n turns (5.4A)

-rigid, right-handed structure

-3.6 residues/turn

-N-H bond of the nth residue points along the helix towards the peptide C=O group of the (n-4)th

residue forming a strong H bond

-R groups project outwards and backwards, providing the surface properties (polarity, charge, solubility)

Favours -> Ala, Glu and Lys

Disfavours -> Pro, Gly and Val

310 helix -> 3 residues/turn, pitch of 6A, longer and thinner

Pi helix -> 4.4 residues/turn, pitch of 5.2A, shorter and wider

Beta-Pleated Sheets

-each amide group is involved in 2 H bonds which occurs between neighbouring polypeptide chains

-chains can be antiparallel (stronger with straight H bonds) or parallel (weaker, H bonds aren’t straight)

-average length of 6 AA (max 15) and rise is 3.5A/residue; strand length would be 6x3.5=21A

-right-handed twist minimizes the steric interactions between residues

Beta-Bends

-non-repetitive, yet well defined

-usually 4 AA to allow sharp turns in the chain

-Gly (flexible), Pro (correct geometry) and Asn are frequent

-often a site of glycosylation of the surface of proteins

Omega loops -> large groups of different, well-defined structures, often involved in protein function, 6-

16AA

Random coil -> non-repetitive structure, no clear pattern

Fibrous Proteins

-protective, connective or supportive role

-insoluble elongated molecule

a-keratins -> 310 residue polypeptide chain

-protofilament (staggered and antiparallel dimers); protofibril (dimer of protofilament);

microfibril (4 protofibrils)

Coiled coil -> 7 AA pseudorepeat; 1 and 4 are nonpolar for favorable packing; rich in Cys residues (forms

Disulfide bonds between chains, cleaved by mercaptans)

Silk fibroin -> -antiparallel B sheet, stacked

-6 residue repeats of Gly-Ser-Gly-Ala-Gly-Ala

-contains regions rich in bulky residues like Arg, Tyr and Val which have less favorable packing,

allowing for flexible regions

Collagen -> makes up connective tissues

-mainly 3-residue repeats of Gly-Pro-Hyp (gly on inside)

-forms long left-handed a-helices that assemble into a right handed triple helix

-Hyp allows for additional H bonding

-deficiency in ascorbic acid (vitamin C) -> absence of Hyp -> scurvy

-His + Allysine – extensive cross-linking from desmosine

-degree of cross-linking increases with age, collagen is more rigid

-Ehlers-Danlos syndrome is the hyperextensibility of skin and joints; osteogenesis imperfect

causes bone fragility, abnormal skin and teeth

Elastin -> very stretchable, found in ligaments, arterial walls, structural proteins of lungs

-rich in Gly, Ala, Val and Pro

-forms random coil chains linked by desmosine, a derivative of Lys

-links a 2,3,4-chain to form a 3D network of fibres

Globular Proteins

x-ray crystallography -> xrays interact with e- clouds, forms e- density maps

limitations -> need to obtain diffracting crystals, static structure, calculation of e- density map from

diffraction pattern requires wavelength of incident light, amplitude of scattered x-rays, phase of the

diffraction and knowledge of the AA sequence

crystal ---(xrays)---- ----(phases)--- - density map ----(fitting)- tomic model

Nuclear Magnetic Resonance -> use radio pulse to expose nuclei to a strong magnetic field so energy

levels split

-monitor the magnetic resonance to frequency of each which is influenced by nearby e-

-COSY (correlation spectroscopy) detects sets of proteins interacting through bonds

-NOESY (nuclear overhauser effect spectroscopy) signals produced by magnetic interactions between

nuclei in close proximity in 3D space but not associated by bonds

Advantages -> don’t require crystals, dynamic, aqueous environment

Disadvantages -> proteins must be <30,000 MW, proteins must be soluble and stable, high conc. Of

protein is required

Tertiary structure -> hydrophobic AA on inside, hydrophilic and charged on the outside, neutral either

BaB motifs – helix connecting 2 parallel B sheets

B hairpin motif – antiparallel strands connected by tight reverse turns

Aa motif – 2 antiparallel a helices packed against each other

Greek key motif – B hairpin is folded over to form a 4-stranded B sheet

a-domains – 4 helix bundles ex. Human growth hormone

a/b proteins – mixtures of helices and sheets ex. Lactate dehydrogenase, carboxypeptidase

B-domains – B barrels ex. Immunoglobulin, retinol binding protein; rolled up 8 B sheets

a/B barrels – triose phosphate isomerase; set of overlapping BaB motifs

domains – polypeptide chains containing more than 200 residues that fold into 2+ globular clusters

Rossmann fold – two BaBaB units combined

Structural Bioinformatics -> how macromolecules are displayed and compared

1. Protein Data Bank – depository for atomic coordinates of proteins; lists the Cartesian

coordinates for each atom or heteroatom; can be viewed using molecular graphics programs

2. Nucleic Acid Database – atomic coordinates of nucleic acids

3. Secondary Databases – structures are more highly conserved than primary sequences; can be

used to reveal distant evolutionary relationships not found by sequence comparison

Quaternary Structure -> different tertiary structures will associate; held together by noncovalent forms,

usually in a symmetrical way

Oligomers – proteins with more than one subunit

Protomers – identical subunits

Cyclic symmetry -> protomers are related by a single axis of rotation (C2,C3,C5)

Dihedral symmetry -> an n-fold rotation axis intersects a 2-fold rotation axis at right angles; an oligomer

with Dn symmetry consists of 2n protomers

Protein Stability

A. Noncovalent forces contributing to folding of proteins

a. Electrostatic forces- much stronger inside a protein, contribute little to stability due to

competing interactions with water; a-helix N terminus is capped by negatively charged AA like Glu or

Asp and the C terminus is capped by positively charged AA like Arg

b. Dipole interactions – energy depends more strongly on distance and orientation than for ionic

interactions

c. London Dispersion Forces

d. Hydrophobic effect

e. Disulfide Bonds

Protein stability (strongest -> weakest): Hydrophobic effect -> H bonding -> ionic interactions -> cross

links (disulfide bonds and inorganic ions)

Zn finger – 2 antiparallel B strands and an a-helix, important in nucleic acid interactions and

transcriptional activators or repressors

Denaturation of proteins -> pH changes, high temperatures, exposure to organic solvents, treatment

with chaotropic agents like urea and guanidium ions which disrupt the hydrophobic effect, exposure to

detergents, reducing/oxidizing agents which disrupt disulfide bonds

Arfinsen Experiment

-proves that protein folding depends on the primary structure

1. 8M urea denatures the protein; mercaptoethanol cleaves its 4 disulfide bonds

2. removal of urea and mercaptoethanol allows protein to renature and re-form disulfide bonds in the

presence of oxygen

3. removal of only mercaptoethanol allows disulfide bonds to form

4. subsequent removal of urea generates an enzymatically inactive protein in which the disulfide bonds

have randomly formed

5. adding a small amount of mercaptoethanol in the absence of oxygen catalyzes the conversion to the

active enzyme through it’s disulfide interchange, allows the native disulfide bods to reform

Determinants in Protein Folding

1. Helices and sheets dominate

2. Directed by internal residues (hydrophobic)

3. Hierarchial organization (one-directional)

4. Protein structure are adaptable –mutations can be accommodated without major change in

structure

5. Context-dependent effects can be important – info specifying secondary structures can be

nonlocal

Folding Pathways

-monitor the rate of folding using:

1. spectroscopy

a. circular dichroism – spectrum of a protein is indicative of its conformation

b. fluorescence – fluorescence of Trp decreases in aq. Solutions

2. pulsed hydrogen exchange – weakly acidic protons found on amine and OH groups exchange with

surrounding water, monitored using 2D NMR spectroscopy

Random coil -> secondary structures -> molten globule state (mostly secondary, some tertiary) -> 3D

structure

-tertiary structure is predicted from sequence of a closely related protein

Folding Accessory Proteins

1. Protein Disulfide Isomerase (PDI)– enzyme which catalyzes thiol-disulfide interchange

2. Peptidyl prolyl cis-trans isomerase (PPI)– cyclophilin is inhibited by cyclosporine A which is an

immunosuppressant to treat patients with organ transplants

3. Chaperones – provide an isolated environment to aid in proper protein folding

a. Heat shock proteins

b. Chaperones – large central cavity to prevent aggregation

c. Hsp 70 –with cochaperone Hsp 40 it unfolds proteins in preparation for membrane transport;

Hsp 90 folds proteins involved with signal transduction

GroEL/ES is an ATP dependent heptamer with C7 symmetry

1. GroEL with 7ATP bound and protein substrate bind to GroES

-conformational change in the GroEL ring, substrate protein starts folding

2. protein folding begins, 7ATP are hydrolyzed, weakened interaction between GroEL and GroES

3. 2nd substrate protein and 7ATP bind to trans ring

4. induces cis ring to release GroES, 7 ADP and better-folded protein substrate

-leaves ATP and substrate protein bound only to the trans ring of GroEL to now become the cis ring as it

binds to GroES

-requires approx. 24 folding cycle for a protein to achieve its native state (7x24=168 ATP)

Protein Misfolding diseases -> Alzheimer’s (amyloid deposits) and Mad cow (prions)

Amyloidases – amyloid is insoluble protein aggregates that interfere with normal cell function,

composed of mainly B sheets

Prion – proteinaceous particles that lack nuclei; PrP prion protein is mainly hydrophobic,

partially proteolyzed

Chapter 7: Myoglobin, Hemoglobin, Muscle Contraction and Antibodies

Myoglobin

-found in skeletal and heart muscles that stores oxygen

-single chain of AA and a heme cofactor folded into 8 helices liked by turns and coils

-the Fe(II) atom at the center of the heme is coordinated by the 4 porphyrin N atoms and one N from His

side chain

-heme is the oxygen binding site that binds to Fe atom and is held in place by hydrophobic interactions

-CO binds 25 000x better than oxygen for free heme-Fe ligand

-binding of oxygen is described by fractional saturation Y

Y = oxygen binding sites occupied/total number of sites= pO2/PO2 + K

-when Y =0.5, pO2 = K

-a hyperbolic curve indicates that the ligand interacts independently with binding sites

Hill Plot

Log(Y/1-Y) = n log pO2 – log p50

-if slope differs from 1, it suggests cooperativity

Hemocyanins – pair of Cu atoms liganded by 3 His

Hemoglobin

-P50 for 4th oxygen bound is 100 fold greater affinity than the 1st oxygen bound

-similar tertiary structure to myoglobin

-[BPG] increases at higher altitudes when pO2 is decreased

-made up of 2a + 2B -> a2B2 held together by hydrophobic effect, H bonds, salt bridges

-functions to transport oxygen from lungs -> muscles and transports CO2, H+ and NO

-from T <-> R transition, a1B1 rotation 15degrees with respect to a2B2 dimer

-T state has more salt bridges than R state and the size of the cavity is larger

-a1B2 (a2B1) move up to 6A relative to B2 upon binding

-a1a2 contacts only in T state where salt bridges stabilize

-B1B2 have no direct direct; the large gap in T state allows binding of BPG

-BPG is an allosteric modulator which influences further ligand binding

-H bonds and salt bridges at the a1B2 interface must be ruptured in the T -> R transition

-as [CO2] increases, affinity of Hb for oxygen decreases

-H+ stabilizes deoxy Hb, favoured at lower pH

Conformational T <-> R Change

1. In T state the Fe(II) in each heme is situated 0.6A out of the heme plane

-oxygen binding shortens the Fe-N bonds by 0.1A causing the porphyrin doming to subside

-Fe(II) moves into the center of the heme plane

2. F helix tilts by 1A across the heme plane so Fe(II) can move into the center of the heme plane

3. causes changes in the tertiary structure, in the T state His 97 in the B chain contacts Thr 41 in the a

hain; in the R state His 97 contacts Thr 38

4. ion pairs are broken from the T-> R transition since they stabilize the T state

Fetal Hemoglobin -> His 143 is replaced by Ser to make a gamma chain, central cavity has fewer +

charges and affinity for BPG is reduced

Allosteric regulation -> ligand at a specific site is affected by the binding of another ligand

a. Homotrophic effect – ligands are identical (ex. Oxygen)

b. Heterotrophic effect – ligands are different (ex. BPG)

Symmetry model -> only the conformational change alters the affinity of a protomer for a ligand;

symmetry is conserved during conformational change, exists in either T or R state

Sequential model -> ligand binding induces a conformational change in a subunit; cooperative

interactions arise via conformational changes on neighboring subunits

-in reality, Hb is a mixture of both models; the quaternary structure is concerted (symmetrical) but

ligand-biding to the T causes small tertiary changes (sequential)

Sickle cell anemia -> Glu6 changed to Val6; HbS fibres precipitate inside the cell and form a sickle shape;

hydroxyurea is used as treatment to increase fetal Hb conc.

Muscle Contraction

Myosin -> 6 polypeptide chains, long rod with 2 globular heads ; a-helical tail consists of coiled coil; head

is an ATPase that bridges to thin filaments

Actin -> G-actin is monomeric, binding sites for ATP, Ca2+ or Mg2+

-F-actin is a double-stranded helix

Tropomyosin -> a-helical homodimer in the grooves of F-actin

Troponin -> TnC binds Ca2+, TnI binds actin, TnT binds tropomyosin

-tropomyosin-troponin complex controls access of myosin to the binding sites on actin

a-actinin -> cross-links F-actin filament, attaches oppositely oriented thin filaments to Z disk

titin -> associate with each thick filament to keep them centered on sarcomere

nebulin -> acts as a template for actin polyermization (tropomodulin caps the – end and CapZ caps the +

end of F-actin)

dystrophin -> anchor F-actin to extracellular matrix, protecting the plasma membrane from being turn

during contraction

-release of Ca2+ from nerves changes troponin C conformation which exposes site on actin for binding

of myosin head

Antibodies

-immunoglobulins consist of 2 heavy and 2 light chains

IgG -> can be cleaved by papain into 2 Fab (arm) fragments and one Fc fragment (body)

-variable regions (VL) and a constant region (CL); heavy chains also have a variable region (VH) and

constant regions (CH1, CH2 and CH3 which are homologous to each other and CL)

Chapter 11: Enzymes

Catalysts – milder rxn conditios, highly specific for their reactants, highly stereospecific

Regulation – allosteric control, covalent modification, enzyme synthesis and degradation, location

Active site consists of: binding site (AA which come into contact with substrate) and catalytic site

(residues responsible for catalysis)

Apoenzyme (inactive) + cofactor -> holoenzyme (active)

Oxidoreductase Transfer or e- or protons Ketone -> alcohol

Transferase Transfer of functional group Phosphate transfer

Hydrolase Hydrolysis Amine -> acid + amine

Lyase Addition of FG to double bonds

Isomerase Isomerization L <-> D

Ligase Bond formation C-O; C-C; C-S

Noncovalent forces involved in binding: van der waals, electrostatic forces, H bonding, hydrophobic

forces

-complementarity between enzyme and substrate based on electronic (charge) and geometric (shape)

-the larger the difference between the free energy of the transition state and that of the reactants, the

less stable the transition state and thus the slower the reaction

-the slowest reaction step is the rate-determining step

-catalysts do not change Keq but they do accelerate the rate the reaction approaches equilibrium

Catalytic Mechanisms

1. Acid-base catalysis – proton transfer between enzyme and substrate

Ex. RNase A catalyzed hydrolysis of RNA

1. His 12 acts as a base promoting nucleophilic attack on the adjacent phosphate and His 119 acts

as an acid and promotes bond breakage

2. 2’3’ intermediate is hydrolyzed; His 12 acts as an acid and His 119 acts as a base

pH optimum factors -> substrate binding, ionization states of AA, ionization of substrates, protein

structure

2) Covalent Catalysis

-the enzyme’s nucleophilic group reacts with electrophilic group on the substrate to form a covalent

bond

3) Metal Ion Catalysis

-binds and orients substrates for reactions, mediates oxidation-reduction reactions, electrostatically

stabilizes or shields negative charges

Ex. Carbonic anhydrase – Zn2+ polarizes water molecule that ionizes to form hydroxyl ion acting as a

nucleophile in the reaction

2. Electrostatic Catalysis

-binding of substrate excludes water from the active site

-charge distribution around the actives sites of enzymes are arranged to stabilize the transition states

5. Proximity and Orientation Effects

-reactions must come together with proper spatial orientation for a reaction to occur

-enzyme brings substrate into contact with their catalytic groups

-electrostatic charged groups help stabilize transition state or guide polar substrates

6. Preferential Binding of the Transition State Complex

-interactions that preferentially bind the transition state

-transition state analogs are stable molecules that geometrically and electronically resemble the

transition state ex. Proline racemase

Lysozyme

-can fit 6 alternating residues of NAG and NAM into the active site

1. lysozyme binds to the hexasaccharide unit in cell wall

2. Glu 35 acts as a general acid catalyst

3. D ring (half chair; NAM) oxonium ion is stabilized by Asp 52

4. Asp 52 nucleophile attaches to C1 of D ring (covalent catalysis)

5. water replaces the E-ring (5th residue) product in active site next to covalent bond

6. Glu 35 assists in hydrolysis of covalent bod by general base catalysis

Serine Proteases

-digestive enzymes with Ser in the active site

-catalytic triad of Asp 102, Ser 195 and His 57

Chymotrypsin -> 2 Gly and Ser allows entry of Phe, Trp and Tyr in active site

Trypsin -> 2 Gly and Asp allows Arg and Lys in active site

Elastase -> Val and Thr allows Ala, Gly and Val in active site

1. Ser 195 nucleophilically attacks the peptide bond to form a tetrahedral intermediate which

involves a transfer of a proton to His 57; aided by Asp 102 which is H bonded to His 57

2. The tetrahedral intermediate decomposes to an acyl-enzyme intermediate from the force of

proton donation from His 57 (general acid catalysis)

3. The amine leaving group is released and replaced by water

4. The acyl intermediate adds water, yielding a 2nd tetrahedral intermediate

5. Reversal of step 1 yield the carboxylate product, regenerating the active enzyme (water is the

nucleophile and Ser 195 is the leaving group)

1. The conformational distortion by the formation of the tetrahedral intermediate causes the

anionic carbonyl oxygen to form an oxyanion hole in the active site which then forms 2 H bonds

with the enzyme

2. The tetrahedral distortion permits the formation of an H bond between the enzyme and the NH

group of the preceding residue of the substrate

-the preferential binding of the tetrahedral intermediate over the acyl-enzyme intermediate is

responsible for the catalytic efficiency of serine proteases

Zymogens

-larger inactive precursors of proteolytic enzymes

Trypsinogen -> trypsin (breakage of Lys15-Ile16 bond)

-autocatalytic activation

-some factors in blood coagulation like prothrombin and fibrinogen are activated by bond cleavage

-hemophiliacs are often missing factor VIII (8) from thrombin

Chapter 12: Enzyme Kinetics

Reaction rates are affected by -> substrate conc., enzyme conc., pH, temperature, inhibitors and

activators

Steady state assumption: ES remains constant until all of the substrate is converted to product

KM -> measure of the efficiency that an enzyme binds to its substrate and converts it to a product

Kcat = Vmax/[E]T is the number of cycles each active site undergoes per unit time (turnover rate)

Kcat/KM is the catalytic efficiency; represents an enzyme’s ability to convert substrate into product

-diffusion controlled limit of 109 M-1s-1

Superoxide dismutase -> arrangement of charged groups on enzyme surface to electrostatically guide

the negatively charged oxygen substrate; binding site is between Cu2+ ion and Arg 143

Other Types of Enzymatic Reactions

1. Multisubstrate – order of substrate binding, different KM values for each substrate

2. Multistep reactions – rate constants of independent steps can be measured using rapid kinetic

procedures

3. Nonhyperbolic reactions – cooperative behavior due to one substrate affecting catalytic activity

of the enzyme on a second substrate

Chapter 12: Part 2 – Enzyme Inhibition

1. Irreversible Inhibition

-any enzyme that can covalently modify an AA side chain in a protein; can act as an irreversible

inhibitor

-suicide inhibitors undergo only a partial reaction and often irreversibly modify the active site

2. Competitive Inhibition

-inhibitor competes directly with normal substrate for binding site

-inhibitor may increase the enzyme’s Km but does affect Kcat or Vmax

Vo = Vmax[S]/aKm + [S]

-acts by reducing the conc. Of free enzyme available for substrate binding

A=1+[I]/K1

K1=[E][I]/[EI]

-apparent Vmax is monitored; determined in the presence of the inhibitor

Methanol poisoning -> need to increase the conc. Of ethanol substrate to overcome the toxic effects

of methanol; competes for alcohol dehydrogenase active site

3. Uncompetitive Inhibition

-inhibitor reacts with enzyme-substrate complex but not to the free enzyme

Vo=Vmax*S+/Km+a’*S+

-effects on Vmax are not reversed by increasing the substrate conc.

4. Mixed Inhibitors

-binds to site other than the active site

-apparent Vmax decreases and apparent Km increases/decreases

-adding more substrate does not reverse inhibition

Vo=Vmax*S+/aKm + a’*S+

Regulation of Enzyme Activities

Aspartate Transcarbamoylase (ATCase) -> E.coli enzyme using Asp and carbamoyl phosphate as

substrates

-homotrophic cooperative binding for both substrates

-regulates pyrimidine synthesis

-example of feedback inhibition, aspartate is the limiting substrate

-inhibited by CTP (homotropic) and activated by ATP (heterotrophic)

-when biosynthesis of purines is high, CTP dissociates from ATCase activating it to produce more

pyrimidines

ATCase structure -> 30k Da, 6 catalytic and 6 regulatory subunits (2 catalytic trimmers (C3) and 3

regulatory dimers (R2))

-R state has a high substrate affinity, prefers ATP while the T state has low substrate affinity, prefers

CTP

-T-> R transition the catalytic trimmers separate along 3 fold axis of symmetry and the regulatory

dimers rotate 15 deg clockwise

-binding of substrate to one subunit increases the binding of other subunits (asymmetrical model)

-binding of both substrates to one subunit induces active site closure, brining both substrates

together

Mechanism for Regulating Enzyme Activity

1. Synthesis/degradation

2. Subcellular location

3. Ionic signal (pH, Ca2+)

4. Covalent modification

Ex. Glycogen phosphorylase -> glycogen(n) + Pi <-> glycogen(n-1) + G1P

-phosphorylation of Ser14 promotes T->R conformational change; acts as an allosteric effector

-phosphorylase b -> ATP + G6P prefer T state and inactive the enzyme while AMP binds to R state to

activate it

-phosphorylase a -> inactivated by excess glucose

Steady-state kinetics -> 1. Determine the binding affinities of substrates, inhibitors and max catalytic

role of the enzyme established

2. Monitoring reaction rates with various conditions to determine catalytic mechanism

3. Gain understanding of the rate of the enzyme in overall metabolic process

4. Enzyme assays to determine the enzyme conc.

Lead compounds = drug candidates with the desired effects

Clinical Trials

-take 7-10 years, tested on humans after animals

Phase I – 20-100 voluntees, determines safety, mode of delivery, dosage

Phase II – 100-500, optimize dosage, drug/placebo

Phase III – confirm efficacy, 1000-5000 people

Cytochrome P450 -> detoxifies xenobiotics, metabolic clearance, steroid synthesis, contains heme,

monooxygenase reaction; converts xenobiotics into water soluble form (toxic compounds)

RH (PAHs, PCBs) + O2 + 2H+ e- -> ROH + H2O

Ex. Tylenol (acetaminophen) -> acetimidoquinone (fatal hepatotoxicity)

Vioxx -> relieves arthritis, pain

-cycolooxygenase-2 selective non-steroidal anti-inflammatory drug

-COX-2 inhibitors block formation of prostaglandins and thromboxanes from arachidonate by

inhibiting the enzyme cycloogenase

Chapter 8: Carbohydrates

-oxidation of polysaccharides yields energy

-play a structural and protective role

-glycoproteins and glycolipids signal location and metabolic fate

Monosaccharides ->

R/D=clockwise

S/L=counterclockwise

Triose=3C, tetrose=4C, hexose=6C (aldohexose and ketohexose)

D-glucose is the only aldose commonly found as a monosaccharide; other sugars include: D-mannose, D-

galactose and D-fructose

Pyranose: Furanose:

-OHs are in equatorial positions except at anomeric carbons where they can be equatorial or axial

-treatment of glucose with a dilute base gives a mix of 3 sugars

-alpha glucose has a mp=146C and [a]=+112.2; beta glucose has a mp=150C and [a]=+18.7

-when either form is dissolved in water, [a] changes to +52.7 due to mutorotation; equilibrium of the 2

hemiacetals resulting in 63.6% beta and 36.4% alpha

Other Monosaccharides

1. Amino Sugars

2. Deoxy Sugars

-OH replaced by H, phosphate, sulphate, etc.

Sugars modified by oxidation/reducation -> ribitol, xylitol, glycerol

Glycoside Bond Formation

-reaction at the anomeric carbon with alcohol

-glycoside bods hydrolyze slowly; no free conversion between a and B forms

Methylation with Methyl Iodide (CH3I)

-premethylation with excess CH3I modifies all H from OH into CH3 groups

Oxidation of the Aldehyde

-easily oxidized to carboxylic acid, used as a test for reducing sugars (sugars containing an aldehyde

or hemiacetal)

1. Copper Oxidation (Benedict’s Test)

R-CHO + Cu2+ -> Cu2O (red, cuprous oxide) + R-CO2H

2. Silver oxidation (Tollen’s reagent)

R-CHO + 2Ag2+(NH3)2 + 2OH -> R-CO2H + Ag + 2NH3

Dissacharides

-1-4, 1-2, 1-3, 1-6 linkages can be formed

1. Maltose

-2 glucoses joined 1-4, reducing sugar b/c of mutorotation around the anomeric carbon

2.Sucrose

-glucose and fructose joined 1-2; inverted sugar (both anomeric carbons involved in bonding)and is not

reducing; easily hydrolyzed

3. Lactose

-glucose and galactose, joined 1-4, reducing sugar, hydrolyzed by lactase

4. Cellobiose

-B-linkages are more resistant to hydrolysis than a-linkages; 2 glucoses joined by B(1-4) linkages,

reducing sugar

-Beta common for structure, alpha for storage

Artificial sweeteners -> saccharin (350x sweeter than sucrose), acelsulfame (180x), aspartame (200x)

Polysaccharides (Glycans)

=polymers of monosaccharides linked by glycosidic bonds

Glucans=polymers of glucose

Galactans=polymers of galactose

Glycoprotein=polymer of AA attached to carbohydrates

Proteoglycan=polymer of carbohydrates attached to protein

A. Storage Polysaccharides

1. Starch

-principal plant food reserve; insoluble granules in plant cytoplasm; composed of a-amylose and

amylopectin

a-amylose -> linear glucose a(1-4) polymer, tends to form left-handed helix of 6 residues/turn

amylopectin -> a(1-4) linkages of glucose, branched with a(1-6) linkages every 25 residues

-starch takes up less space than glucose; can be readily hydrolyzed due to alpha linkages and

branching

-cleaved randomly by a-amylase in saliva to produce glucose and in small intestine to produce

maltose, maltotriose and dextrins (oligosaccharides with a1-6 linkages)

-hydrolases further degrade starch

-in polymeric form , glucose greatly reduce osmotic pressure

2. Glycogen

-storage polysaccharide of animals

-structure resembles amylopectin but is more compact; branches every 8-12 residues; can be rapidly

broke down

-degraded by glycogen phosphorylase to produce G1P

3. Dextrans

-storage and structural polysaccharide of yeast and bacteria

-glucose monomers linked a(1-6) with a(1-3) branches

-different size polymers are used for making chromatography gels, must be run in ethanol or the

bacteria will digest dextran

B. Structural Polysaccharides

1. Cellulose

-most abundant biopolymer, structural component of plants

-linear polymer of glucose B(1-4) linkages

-B-linkages produce extended, planar structure; stabilized by H bonds

-only certain herbivores (ruminants) have cellulases capable of hydrolyzing the linkages

2. Chitin

-structural unit of the exoskeleton of invertebrates, fungi and algae in cell walls

-polymer of N-acetyl-D-glucosamine residues linked B(1-4)

3. Glycosaminoglycans

-gel-like substance in extracellular matrix in cartilage, tendon, skin, eyes

-linear, heteropoysaccharide (more than one type of sugar)

-repeating disaccharide of negative sugar and hexosamine side chains

-lubricates joints and vitreous humor in the eye

-high negative charge density allows extended conformation, high viscosity, elasticity

Heparin -> injectable anticoagulant, has highest negative charge density, found in intracellular mast

cells lining arterial cell walls

-binds to the enzyme-inhibitor antithrombin causing a conformational change

-the activated ATI-III inactivates thrombin and other proteases involved in blood clotting, including

factor Xa -> blood thinner

Tissue Plasminogen Activator (tPa)

-a serine protease found on surface of endothelial cells of veins, capillaries, pulmonary artery, heart

and uterus; secreted after injury

-plays a role in cell migration and tissue remodeling; increased activity results in excessive bleeding

-tPa is a thrombolytic agent – can dissolve blood clots

Biofilms -> form when bacteria adhere to surfaces in aqueous environments and begin to excrete a

slimy substance that can anchor them to surfaces; bacteria as biofilms are better able to defend

themselves against immune cells

Glycoproteins

A. Proteoglycans

-core protein with 1 or more linked glycosaminoglycan side chain

-found in ground substances, extracellular matrix, organize tissue morphology; heavily glycosylated

glycoproteins

B. Bacterial Cell Walls

-penicillin inactivates enzyme that cross-links peptidoglycans

-discovered by Sir Alexander Fleming

-can be converted into penicillinoic acid by penicillinase

-inhibits transpeptidation (cross-linking) of the cell wall by binding to D-Ala-D-Ala sequence in

peptidoglycan

-B-lactamase-resistant semi-synthetic penicillins like methicillin were developed

C. Glycoprotein Structure and Function

-1/3 of all mammalian proteins contain a covalently linked saccharide

-carbohydrates mediate interprotein and intercellular interactions

-variable chain length and site of attachment of sugar onto the protein is microheterogeneity: a

given protein occurs in different glycosylated forms

Carbohydrates can be..

1. O-linked to Ser or Thr

-almost all of the secreted and membrane-associated proteins in eukaryotic cells are glycosylated

-protective functions; clustered segments with extended conformations, mucins-protein components of

mucus (lubrication)

-composed of large O-liked glycoproteins

-eukaryotic cells have a fuzzy coating of o-linked glycoproteins and glycolipids called the glycocalyx

2. N-linked to Asn side chain

-N-linked glycoproteins exhibit numerous glycoforms; differ in sequences, locations and number of

covalently attached oligosaccharides

-no generalizations can be made about the effects of glycosylation on protein properties

-carbohydrates on cell surface were less numerous in cancerous cells

-carbohydrates are added post-translationally and processed by several enzymes

Carbohydrate Analysis

1. Affinity chromatography – immobilizes sugar-binding proteins (lectins) onto resin

2. Methylation analysis – methylate oligosaccharide with CH3I, acid hydrolysis cleaves glycosidic

bond, analyze sugars by reducing sugar test, chromatography, mass spectrometry to determine

which carbon is involved in glycosidic bod

3. Periodate treatment – determines aldose vs ketose

4. Exoglycosidases – hydrolyze specific monosaccharide from non-reducing end then analyze via

affinity chromatography or methylation

5. Endoglycosidases – cleave between non-terminal sugars ex. Lysozyme

6. NMR

Functions of Oligosaccharides on Proteins

1. Structural – shield proteins surface, modify activity, restrict conformational freedom, protein

sorting, direct protein to final destination

2. Recognition – intracellular communication, cell-cell recognition (lectins-proteins that bind

carbohydrates)

3. Antigenic determinants – ABO blood antigens; oligosaccharide components of glycoproteins and

glycolipids on cell surface

O type – H antigen; A type – A enzyme (glycosyltransferase) adds the A-antige (a1-3 N-

acetylgalactosamine); B type – B enzyme, adds the B antigen (a1-3 galactose)

Xenotransplants -> using animal organs to save humans; cell surface galactosyl-a(1-3)-galactose

disaccharides not found in humans

Chapter 9: Lipids and Biological Membranes

A. Fatty Acids

-carboxylic acids with long hydrocarbon chains

-insoluble in water but soluble in organic solvents (CHCI3)

-first double bond often occurs at position 9

Saturated Fatty Acids

12:0 Lauric Acid dodecanoate (12 carbons, 0 double bonds)

16:0 Plamitic Acid hexadecanoate (16 carbons, 0 double bonds)

18:0 Stearic Acid octadecanoate (18 carbons, 0 double bonds)

Unsaturated Fatty Acids

18:1n-9 Oleic Acid (18 carbons, 1 double bond 9 away from the last carbon)

18:2n-6 Linoleic Acid (18 carbons, 2 double bonds, last one is 6 away from terminal methyl group) ex. Of

omega-6 fatty acid

-mp increases when the number of carbons increases

-saturated fatty acids are highly flexible – fully extended to minimize the steric interference of CH2

groups

-mp decreases as the number of double bonds increases; kinks cause less compact packing

B. Triglycerides

-energy reservoirs in animals yield a lot of energy upon oxidation

-esters of fatty acids and glycerols stored in cells as adipocytes

-mp is determined by fatty acid content; very hydrophobic

-can be hydrolyzed in boiling water in the presence of acid, base or lipases

-saponification (NaOH) -> soaps + glycerols

-vegetable oils are triglycerides with 80% unsaturated fatty acids (liquid at RT) while animal fats are

saturated fatty acids (solid at RT)

Hydrogenation -> controls the shape, texture and shelf-life of food; addition of H molecules

Trans fats -> vegetable oils are heated and exposed to H2, changes configuration from cis to trans,

increase LDL and reduce HDL in human body, olestra (6-8 fatty acids+sucrose) inhibits the absorption of

some vitamins and nutrients

B. Glycerophospholipids

-form the bulk of all membranes

-nonpolar tails and polar heads

-can be cleaved by phosholipases

-phospholipases disrupt cell membranes, lysing cells; found in bee and snake venom

-glycerophospholipids can be hydrolyzed by strong acids/bases; results in glycerol, free fatty acids

and alcohol

D. Sphingolipids

-major membrane component

-ceramide is an N-acy fatty acid derivative of sphingosine

1. Sphingomyelins – sphingophospholipids found in plasma membranes, myelin sheath where

X=phosphocholine or phosphatidylcholine

2. Cerebrosides – sphingoglycolipids (ceramide+sugar); no phosphate, found in neuronal cell

membranes (x=galactose) or nonneuronal cell membranes (x=glucose, called a

glucosylcerebroside)

3. Gangliosides – similar to cerebroside except x=oligosaccharide; terminal sugar is usually sialic

acid

-components of cell surface membranes; oligosaccharide extends beyond cell surface; functions as

receptors for hormones and toxins

-important for cell-cell recognition in growth, differentiation, immune response

Tay-Sachs -> accumulation of partially degraded ganglioside in brain; missing hexoaminidase A which

cleaves off extra sugar in GM3-GM2

Arachidonic acid -> NSAIDs like aspirin and ibuprofen block the formation of prostaglandins and

thromboxanes from arachidonic acid, a precursor to eicosanoids

-eicosanoids are paracrine hormones that act only on cells near the point of synthesis

-prostaglandins regulated 3’5’-cyclic AMP (smooth muscle contraction) while thromboxanes regulate

blood clotting

-leukotrienes induce contraction of muscle lining airways to lungs – overproduction leads to asthmatic

attack

E. Non-Saponifiable Lipids

1. Terpenes – vitamin A (retinol)

2. Steroids – cholesterol (vitamin D)

-testosterone is produced in testes while estradiol is produced in ovaries and placenta

-cortisol and aldosterone regulate glucose metabolism and salt excretion; synthesized in the cortex of

the adrenal gland

Cell Membranes

-consist of a mixture of lipids (phosphoglycerides and sphingolipids), proteins (glycoproteins) and

carbohydrates (attached to lipids and proteins)

A. Structure of Mono/Bilayers

1. monolayers – form at air-water interface; polar head in water and hydrophobic tail in the air

2. Micelles – CMC (critical micelle concentration); below CMC individual molecules predominate

and above CMC there is spontaneous formation of micelles

3. Bilayers – phosphoglycerides and spingolipids tend to form bilayers

C. Dynamic Character of Phospholipid Bilayers

-lateral diffusion and rotation about its own axis are frequent while flip-flop from one side to the other is

rare

-bilayers can be destroyed by detergents

-they are impermeable to most polar substances but permeable to most hydrophobic substances and

water

-Tm can be 10-40C

-membrane fluidity is controlled partially by packing and order of hydrophobic tail; about 15% more

compact in fluid state

-cholesterol keeps fluidity of the membrane constant through temp. fluctuations and decreases the

mobility of other membrane constituents (ex. Glycoproteins)

Properties of Archaeal and Bacterial Membranes

-liposomes composed of archaeal tetraether lipids are more stable than those of bacterial bilayers

-phytanyl chains contain methyl groups at every 4th carbon in the backbone making the segmental

motion in the phytanyl chains hindered due to methyl side groups

-longer membrane spanning hydrocarbon chains; ether links are far more resistant to oxidation and high

temps than ester links

-phytanyl side chain is not susceptible to degradation at alkaline pH and degradation by phospholipases

Integral Membrane Proteins

A. Hydropathy Index

-measure of relative polarity of AA

Delta G = free energy of transfer of AA from hydrophobic solvent to water

-positive for aromatic AA (unfavorable) and negative for charged/polar AA (favorable)

Ex. Bacteriorhodopsin -> contains alpha helices, light driven H+ pump, bundle of 7 transmembrane

helical rods

Porins -> contains beta barrels, similar secondary structure to globular proteins

Peripheral proteins – weakly bound to membrane surface mainly through ionic and H bonding; liberated

easily by H bond disrupting agents ex. NaCl, proteases

Myristolation – N-terminal Gly

Palmatoylation – S of Cys; amide link to c-terminal carboxyl

Biological Membrane Structure

-composed of proteins associated with lipid bilayer matrix

Variable lipid composition – ratios of phosphoglycerides to sphingolipids, etc.

Protein: lipid ratio – 0.23(myelin) to 3 (gram+ bacteria)

Experimental proof of fluid mosaic model -> fusion of mouse and human cells, cell-surface markers were

fluorescently labeled

Erythrocyte Membrane

-50% protein, isolated by breaking cells by osmosis, solubilized using SDS, separation by SDS-gel

electrophoresis yields 7 major proteins

1. Spectrin – peripheral protein, forms the inner skeleton of erythrocyte; 2 antiparallel chains (alpha

subunit is 280k Da while B subunit is 246k Da)

-chains are loosely intertwined to form long, flexible dimers, which form a dense, irregular meshwork on

the inside of the membrane

-attaches to actin and ankyrin proteins

2. Ankyrin – binds to integral membrane ion channel protein

-24 tandem (33 residue) repeats

-underlying skeleton of membranes along with cytoskeleton limit the mobility of integral membrane

proteins

3. Anion Channel – co-transport of HCO3-, Cl-

-929aa + 5-8% carbohydrate

-integral protein, 2 subunits with 8 helices each

-approx 1/3 of total membrane proteins

4. Glycophoryn A

-131aa + 60% carbohydrate

The Fluid Mosaic Model

-lipid distribution is asymmetric

-proteins can diffuse only laterally and rotationally; movement is restricted by other protein

components

-fatty acids in the bilayer are usually cis conformation

-glycoproteins and glycolipids are oriented with carbohydrate moieties facing the cell’s exterior

Ex. Glycosphingolipids (outer surface) and cholesterol pack together to form mobile rafts and

indentations called caveolae which associate with specific proteins

Chapter 10: Membrane Transport

Chemical potential difference: difference in the conc. Of the substance on both sides of the membrane,

substance will move down the conc. Gradient unless coupled to an exergonic process like ATP hydrolysis

Membrane potential: electrical potential difference due to charge difference across a membrane

resulting from the transmembrane movement of ions – electrochemical potential

Transport Across the Membrane by Diffusion

-occurs across a permeable divider, down the conc gradient ex. H2O, N2, CH4

-ion movement alters membrane potential, opening of Na+ channel results in action potential,

stimulates opening of additional Na+ channels further along the axon

Transporters (Permeases)

1. Facilitated Diffusion

-requires carrier-protein but no energy is required

-moves ions down the conc gradient, thermodynamically favorable

a. ionophores: carry ions across the membrane

carrier ionophores -> bind to ion, diffuse through membrane, release on other side, ionic

complexes are soluble in nonpolar solvents ex. Valinomycin (K+ ion)

channel-forming ionophores -> transmembrane channels and pores

b. porins – trimmers with each subunit composed of a 16/18 B barrels; not very selective

c. ion channels – highly selective, roles in maintaining osmotic balance, signal transduction,

neurotransmission

uniport – movement of a single molecule at a time

symport – simultaneous movement of 2 molecules, same direction

antiport – simultaneous movement of 2 molecules, opposite directions

d. aquaporins – rapid movement of water but not solutes or ions; 6 transmembrane a-helices

-proton exclusion to prevent electrochemical potential formation

-aromatic/arginine constriction

Glucose Transport in Erythrocytes (GLUTI)

-alternates between conformations to bind and release ligand (glucose) on opposite side of the

membrane

-passive transporter involved in facilitated diffusion

2. Active Transport

-moves against the conc. gradient; requires carrier protein & ATP, reduces Ea and delta G for

transmembrane diffusion, endergonic reactions

-results in build-up of a solute on one side of the membrane

Ex. ATP + water -> ATP + Pi, oxidation reaction, absorption of light

Na+/K+ ATPase Pump

-maintain low [Na+] and high [K+] intracellularly

-pumps 2K+ in and 3Na+ out per ATP

-creates an electrochemical gradient important for: osmotic control, active transport of glucose and AA,

muscle contraction

-controlled by the phosphorylation/dephosphorylation of a specific Asp residue

-ATP phosphorylation of E1 form in the presence of Na+ to form E1-P

-hydrolysis of phosphate in presence of K+ to form E2

-each conformation (E1, E2) have different structures, catalytic activities and ligand specificities

Glucose into cell-uniport

Glucose/Na+ out of cell - symport

Na+/K+ - antiport