Chapters 1, 2 Study Objectives
Polar vs. nonpolar compounds: Be able to differentiate compounds, or portions of molecules according to this characteristic; amphipathic compounds
Dipoles, dipole moment
Electrostatic interactions: example(s); factors contributing to force of electrostatic interaction (an equation); meaning of dielectric constant
distinguish charge-charge, charge-dipole, dipole-dipole
induced dipoles
van der Waals interactions; van der Waals radius
Hydrogen bonds: physical basis, structure of water, directionality
Be able to identify hydrogen bonds and draw potential hydrogen-bonded groups.
Hydrophobic effect: hydrophobic vs. hydrophilic
physical basis for hydrophobic interactions
ionization constant of water (Kw)
pH: mathematical definition; Be able to use this expression
differentiation between strong acids, bases and weak acids, bases - know examples
pKa: definition and meaning
titration curves: understand thoroughly
buffers: meaning, significance on titration curves
Henderson-Hasselbalch equation:
know this; also derivation
Be able to work problems using weak acids, bases and buffers.
Biologically-relevant buffer systems:
phosphate buffer inside cells: Understand multiple dissociation of phosphoric acid and ranking of pKs
histidine buffer system inside cells
bicarbonate buffer system in blood plasma: Understand multiple equilibria involved in this example

Chapter 3 Study Objectives
enthalpy: deltaH = deltaE + PdeltaV (at constant pressure); differentiate internal energy and PV work; exothermic vs. endothermic reactions
meaning of entropy
Gibbs free energy: G = H - TS
deltaG = deltaH - TdeltaS (at constant temp. and pressure)
endergonic vs. exergonic
relationship of deltaG°' and equilibrium constant
differentiation of deltaG°' and deltaG
be able to calculate actual deltaG at any concentrations of reactants and products
van't Hoff plot: mathematical expression and usefulness
structure of ATP: phosphoester bond vs. phosphoanhydride bond
coupling of ATP hydrolysis with endergonic reactions
addition of free energy changes
multiplication of equilibrium constants
phosphate group transfer potential
creatine/creatine phosphate in muscle (creatine kinase)

Chapter 4 Study Objectives
general structure of amino acids; be able to draw in either L- or D- (or R- /S-) configurations
know structures of R-groups for 20 normal amino acids
classification into groups
distinguish hydrophobic/hydrophilic ones
alpha to epsilon nomenclature to distinguish carbon chain
disulfide bond between cysteine sidechains
approx pKs of side groups (which ones ionize: basic, acidic, pK of his = 6; also cys and tyr) -know relative order of pKs
general types of modifications that can occur to R-groups
titration curves of amino acids
- need to be able to draw
- determination of net charge at various pH values
isoelectric point (pI): definition, calculation of
UV absorbance of aromatic amino acids / use of Lambert-Beer law to determine concentration from absorbance
general types of reactions that occur with amino or carboxyl groups
names of reagents that react with cysteine thiol group
formation of Schiff base between aldehyde and amino group
ion-exchange chromatography
- distinguish cation-exchange vs. anion-exchange – examples of each type
- be able to predict order of elution of amino acids by this method
peptides: draw peptide bond; N to C convention of naming peptide chains; examples of biologically-active peptides

 Chapter 5 Study Objectives
Distinguish primary, secondary, tertiary, quaternary levels of protein structure
Ave. formula weight of amino acid in protein = 110 (know how to estimate molecular weight of protein from # of amino acids and vice versa)
total hydrolysis of protein in 6N HCl - amino acid analysis; understand potential uncertainties with this technique
determination of amino acid sequence of proteins - general strategy; first done by F. Sanger with insulin
methods to cleave disulfide bonds: dithiothreitol or beta-mercaptoethanol followed by iodoacetic acid; or, performic acid oxidation
determination of amino-terminal amino using phenylisothiocyanate
determination of carboxy-terminal amino acid - carboxypeptidase
site-specific cleavage of polypeptide chains
trypsin (an enzyme): after carboxyl of arg, lys
chymotrypsin (an enzyme): after carboxyl of phe, trp, tyr
cyanogen bromide (a chemical): after carboxyl of met
Edman degradation, phenylisothiocyanate, PTH-amino acids
ordering of peptide sequences by overlap
mass spectrometry methods: MALDI, ESI, tandem mass spec (MS/MS)
comparison of protein sequences: homologous proteins, invariant residues, conservative substitutions
Protein purification - activity, yield, specific activity, fold-purification, purification table
nonchromatographic protein purification techniques: crude extract, differential centrifugation, selective precipitation by ammonium sulfate, differential thermostability, dialysis
chromatography methods: ion-exchange, gel filtration, affinity, hydrophobic interaction
electrophoresis of proteins
SDS polyacrylamide gels
estimation of molecular weight of individual polypeptide chains
use of reducing agents: beta-mercaptoethanol and dithiothreitol
isoelectric focusing
2D gels combining isoelectric focusing and SDS polyacrylamide gel electrophoresis
sedimentation of proteins - sedimentation coefficient (S)
solid phase synthesis of peptides (B. Merrifield) - understand basic technique; don't need to know chemical details

Chapter 6 Study Objectives
characteristics of peptide bond: planarity, usually trans configuration, electric dipole
phi and psi angles for N-Ca and Ca-C bonds in polypeptide chain
alpha helix - be able to identify on structures
- proposed by Pauling
- right-handed (understand right-hand rule)
- R-groups point outward
- periodicity of 3.6 amino acid residues (per turn)
- pitch of 0.54 nm
- helical wheel depiction (looking down the axis)
- hydrogen bonding in alpha helix
- electric dipole along axis of helix (pos. on N-terminal end and neg. on C-terminal end)
factors affecting stability of alpha helices (be able to apply to a problem)
- helix propensities - gly and pro are bad
- proline is usually not favorable - ring structure constrains rotation of phi angle; no hydrogen on N for hydrogen bonding
- electrostatic repulsion or attraction
- charged side chains at ends of helix stabilize or destabilize depending on electric dipole of helix
Beta sheets - be able to identify on structures
- beta strand
- parallel vs. antiparallel
- pleated structure
- hydrogen bonding between strands
Turns or bends - be able to identify on structures
- "favored" amino acids are gly (because small, no steric interference) and pro (because can be in cis configuration which causes bend in polypeptide chain)
Ramachandran plot - what does it mean?
- specific regions for various types of secondary structure
Fibrous proteins - distinguish properties from globular proteins
alpha-keratin, fibroin
collagen: left-handed helix (not alpha helix); gly-X-Y repeat; 4-hydroxyproline; hydroxylation reaction requires vit. C; tropocollagen - triple helix; collagen cross-linking
X-ray crystallography - general method; diffraction pattern, electron density map, resolution of structures
NMR spectroscopy - 2D NMR
amino acid location in folded proteins
supersecondary structures: alpha-alpha; beta-alpha-beta; beta hairpin; beta barrel; doubly wound parallel beta sheet; beta sandwich
protein domains - protease susceptibility between domains
proteins are only marginally stable (when compared to denatured state)
protein denaturation
heat (melting temperature)
urea, guanidine hydrochloride
Anfinsen expt: denaturation and refolding of ribonuclease (significance)
thermodynamics of protein folding
enthalpic effects (deltaH): internal interactions
conformational entropy (deltaS < 0)
hydrophobic effect (deltaS > 0)
overall deltaG < 0, but small
role of disulfide bonds in protein stability
kinetics of folding - intermediates, molten globule, cis-trans proline isomerization
chaperonins: GroES/GroEL complex in E. coli
protein misfolding diseases - prions, Alzheimer's beta amyloid
immunoglobulin quaternary structure - heavy chains, light chains, disulfide bridges, Fab regions vs. Fc region; Y-shaped structure; immunoglobulin fold (beta sandwich)

Chapter 7 Study Objectives
know structures: glyceraldehyde, dihydroxyacetone, D-ribose (straight-chain and cyclic), D-glucose (straight-chain and cyclic), D-fructose (straight-chain and cyclic), D-mannose (straight-chain and cyclic), D-galactose (straight-chain and cyclic)
aldose vs. ketose
numbering of carbon atoms
stereochemistry: distinguish D- vs. L-, meaning of epimer; distinguish alpha vs. beta configurations in cyclic structures
cyclic monosaccharides: hemiacetal, hemiketal formation; anomers, anomeric carbon, mutarotation; chair conformation; pyranose vs. furanose rings
types of modifications: deoxy, amino, phosphate, oxidized groups (i.e., gluconic acid, glucuronic acid), alditols
reducing vs. nonreducing sugars; Fehling's reaction (reduction of copper to form cuprous oxide)
disaccharides: O-glycosidic bond; be able to interpret or draw structures from descriptive name
polysaccharides: compare and contrast types of linkages, branching, overall structure of starch (amylose, amylopectin), glycogen, cellulose, chitin; reasons for storage of glucose units in polymer instead of monomer subunits
general composition of glycosaminoglycans - names; types and characteristic negative charges
general composition of extracellular matrix: proteoglycans; glycosaminoglycans
glycoproteins: O-linked vs. N-linked carbohydrates - amino acids involved, be able to draw structure of linkages
features of bacterial cell wall: peptidoglycan; degradation by lysozyme
blood group antigens as example of role of cell surface carbohydrates in recognition
- How do different carbohydrate structures lead to blood donation characteristics of A, B, AB and O-types?

Chapter 8 Study Objectives
fatty acids - general structure; numbering scheme; saturated vs. unsaturated; effect of double bonds on structure; effect of length and saturation on melting point
triacylglycerol (triglycerides) - general structure; alkaline hydrolysis to make soaps
waxes - general structure
glycerophospholipids (phosphoglycerides) - general structure; know structures of ethanolamine, choline, serine and how they link to phosphoglyceride structure; recognize structure of inositol
phospholipases - names and cleavage specificities
sphingolipids - recognize structure of sphingosine and know how sphingolipid structure is put together
ceramide, sphingomyelin, cerebrosides, gangliosides
terpenes - know structure of isoprene unit
steroids - steroid ring nucleus; cholesterol: know structure; steroid hormones - examples

Chapter 9 Study Objectives

membrane structures - micelles, lipid bilayer, liposomes
lipid mobility in bilayers - flip-flop not allowed; lateral diffusion is fast
membrane fluidity - transition temperature, effect of cholesterol
effect of temperature change on fatty acid composition of membrane lipids
fluid mosaic model of membranes
thickness of membrane: ~5-7 nm (3 nm for lipid bilayer)
membrane sidedness: different lipid compositions on each leaflet; carbohydrate moieties point to outside
membrane proteins - differentiate properties and methods of extraction
peripheral (extrinsic) proteins
integral (intrinsic) proteins
lipid-linked proteins
hydrophobicity index / hydropathy plots - indicate membrane-spanning regions
transmembrane helices for integral membrane proteins (bacteriorhodopsin example)
beta barrel structure of porins
lipid-linked proteins:
- prenylated proteins - thioether linkage
- fatty-acylated proteins – amide linkage to myristate or thiester linkages to palmitate
- GPI-linked proteins
differentiate types of membrane transport and know examples of each
thermodynamics of transport; be able to calculate free energy changes; role of transmembrane electrical potential
passive diffusion (nonmediated transport)
differentiation of mediated vs. nonmediated transport
facilitated diffusion- pores, carriers, transporters
ionophores: gramicidin A, valinomycin
erythrocyte glucose transporter
erythrocyte anion transporter (bicarbonate exchanged with chloride ion)
uniport vs. symport vs. antiport
active transport
differentiate primary vs. secondary (cotransport)
sodium-potassium pump: stoichiometry of transport, inhibitors, mechanism
(H+-K+) ATPase
osteoclast proton pump
light-driven active transport: bacteriorhodopsin
example of co-transport: sodium-glucose transporter

Chapters 10, 11 Study Objectives
be able to draw structures of adenine, guanine, cytosine, thymine, uracil - know numbering scheme so that can draw simple modified forms too
understand and draw keto-enol and amino-imino tautomerism
be able to draw nucleosides with (N-glycosidic) linkage to ribose or deoxyribose
anti vs. syn conformations
be able to draw nucleotides - distinguish between mono-, di- and tri-phosphates; alpha, beta, gamma phosphate nomenclature
phosphodiester backbone of DNA and RNA
- be able to draw structure
- 5' and 3' ends
- ave. formula weight of mononucleotide is approx. 330 (single-strand of DNA)
Chargaff's rules: A=T, G=C, pyrimidine=purine
be able to draw A-T (or A-U) and G-C base-pairs with correct hydrogen bonds
alkaline hydrolysis of RNA
nucleases - types of specificities: RNase vs. DNase, exonuclease vs. endonuclease, a-type vs. b-type cleavage
restriction enzymes
- restriction-modification (methylation) systems
- properties of class II enzymes
- probability of occurrence of cutting sites
- sticky (overhanging) ends vs. blunt ends after cutting
DNA double helix
- right-handed
- antiparallel strands
- major vs. minor grooves
- stabilization by base-stacking and hydrogen bonds
- distinguish B, A and Z forms - bp/turn, tilting of base-pairs, handedness of helix, natural occurence of each form
intercalating agents: ethidium bromide, acridine orange, actinomycin D
factors that promote or affect denaturation of DNA (be able to predict relative effects of each): heat, ionic strength, chemicals (formamide, urea), pH, length (if duplex is short), base composition (%GC)
melting curve of DNA - hyperchromic effect, melting temperature (Tm; relationship to stability of helix)
hybridization: second-order kinetics; DNA-DNA or DNA-RNA or RNA-RNA (if antiparallel and complementary)
supercoiling of DNA
relaxed vs. positive supercoiling vs. negative supercoiling
linking number (L): L = T + W
- understand what each term means, sign of each term, how L can change
underwound vs. overwound DNA - meaning and significance
superhelical density: sigma
- know equation and what each term means; what is significance of this number
topoisomerases: functions; topo I (nicking-closing enz.) vs. topo II (DNA gyrase) - What are activities of each?
chromatin structure - significance of DNA compaction
- experimental evidence (electron microscopy, micrococcal nuclease repeat pattern of DNA sizes)
- structure of core particle: histone composition, size of DNA
- supercoiling of DNA around outside of histone core
- linker histone (H1)
- compaction of DNA not nearly sufficient (only 7-fold)
higher-order chromatin structure
- highly speculative model of further coiling of DNA
RNA - classification into stable (tRNAs, rRNAs) vs. unstable (mRNAs)
secondary structure of RNAs: stem-loop structures (understand how inverted repeat sequence leads to this structure), intramolecular base-pairing: G-U base-pairs as well as A-T and G-C; A-type double helix
cloverleaf structure of tRNAs
general 3D structure of tRNAs
DNA sequencing techniques
- Sanger (dideoxy, enzymatic, chain termination): understand concepts and order of steps in the process (understand Fig. 11.3)

Chapter 12 Study Objectives

recombinant DNA - definition
types of cloning vectors used in E. coli (bacteria)
- plasmids: properties (understand how these properties are important for function as a cloning vector)
- bacteriophage lambda - advantage of using lambda as a vector instead of plasmid
function of DNA ligase
directional cloning into a plasmid vector
expression vectors
meaning of transformation
DNA microarrays
PCR (polymerase chain reaction)
- understand concepts and steps in the procedure
- significance of this technique
- QuickChange mutagenesis technique

Chapter 13 Study Objectives
enzymes increase rate but no effect on direction of chemical reaction
active site
cofactors and coenzymes
enzyme classification - remember six classes but not numbers; general idea of E.C. classification scheme
reaction profiles - be able to interpret
delta G, transition state, activation energy, deltadeltaGact, enzyme-substrate complex
general relationship between rate constant and activation energy: k = (constant)e(-deltaGact/RT)
rate enhancement: kcat / kuncat
first-order (unimolecular) vs. second-order (bimolecular) reactions
rate constants
rate equations for first-order and second-order (A + A -> P) reactions
derivation of Michaelis-Menten equation
- how to write rate equations
- steady state assumption
- definitions of Km, Vmax
- [Etotal] = [E] + [ES]
v0 vs. [S] hyperbolic curve - determination of Km
use of double-reciprocal (Lineweaver-Burk) plot and Hanes-Woolf plot
meaning of kcat (turnover number)
Vmax = kcat[Etotal]
meaning of kcat / Km - the catalytic efficiency of an enzyme
NSAIDs (non-steroidal anti-inflammatory drugs) as examples of enzyme inhibitors: aspirin, acetaminophen, ibuprofen
- COX (cyclo-oxygenase) inhibitors; COX-1 and COX-2 isoforms; COX-2 inhibitors (Celebrex, Vioxx)
differentiation of types of reversible inhibitors by binding equilibria and double-reciprocal plots
- competitive, noncompetitive: pure vs. mixed inhibition, uncompetitive
KI (dissociation constant for binding of inhibitor to enzyme)
irreversible inhibitors - DIPF; suicide inhibitor (substrate) - penicillin
multisubstrate (bisubstrate) reactions - draw binding equilibria for sequential mechanism (random vs. ordered) vs. double-displacement (ping-pong) mechanism
ribozymes: examples - Tetrahymena self-splicing intron, ribonuclease P, peptidyl transferase activity of ribosomes

Extra Material - Determining Rate Constants
general method to determine dissociation rate constant for AB
how to block reassociation when determining k-1
potential problems when determining k-1
general method to determine association rate constant for AB
k observed, need to have [B]i >> [AB]max
plot k observed vs. [B]i in order to determine k1

Chapter 14 Study Objectives
general principles of enzymatic catalysis
- proximity and orientation
- preferential binding of transition state - relative destabilization of ES complex compared to enzyme-transition state complex
general acid-base catalysis - mechanism of RNase A uses his sidechains as both general acid and general base
covalent catalysis - good nucleophiles in enzymes
metal ion catalysis
mechanism of chymotrypsin (a serine protease)
- other examples of serine proteases - trypsin, elastase
- DIFP as an irreversible inhibitor of active site serine
- catalytic triad
- specificity pocket
- understand roles of general acid-base catalysis and covalent catalysis in mechanism
- acylation and deacylation phases of mechanism - understand chemistry and be able to draw structures
- stabilization of transition state using oxyanion hole of active site
mechanism of aspartic proteases
- examples of aspartic proteases: pepsin, Cathepsin D, Renin, HIV protease
- pH dependence of activity
- two aspartic acid sidechains involved as general acid / general base
- be able to draw (and understand) mechanism
HIV protease
- function in HIV gene expression
- homodimer with each monomer supplying one asp to active site
- example of competitive inhibitor as HIV therapeutic: Crixivan
- function to catalyze hydrolysis of glycosidic linkages in peptidoglycan of bacterial cell wall
- first enzyme with crystal structure in 1965
- substrate binding to active site via hydrogen bonds
- distortion of one glucose residue in substrate at the cleavage site
- general acid/base catalysis with glu residue in active site
- distinction between two possible mechanisms - one with carbonium ion intermediate; another with covalent asp-glycosyl intermediate

Chapter 15 Study Objectives
zymogen or proenzyme - general concept, examples
isozymes - example of lactate dehydrogenase
modulator proteins - example of regulatory subunit of cAMP-dependent protein kinase
feedback inhibition, activation
allosteric enzymes -
- homoallostery (homotropic) vs. heteroallostery (heterotropic effect)
- kinetic treatment with sigmoid curves (not Michaelis-Menten)
- T state vs. R state
- shift of K0.5 with positive vs. negative modulators - K system
- contrast to V system (altered Vmax)
reversible covalent modification of enzymes - phosphorylation-dephosphorylation
example of glycogen phosphorylase
allosteric effectors of glycogen phosphorylase b: activated by AMP, inhibited by ATP, glucose-6-phosphate
cascade of reversible phosphorylation to control glycogen phosphorylase:
- phosphorylase b vs. phosphorylase a
- phosphorylase kinase
- cyclic AMP-dependent protein kinase
- synthesis, degradation of cAMP by adenyl cyclase, phosphodiesterase
- role of G protein in transducing signal from hormone to adenyl cyclase

Extra Material - Determining Affinity Constants for Two Component Binding; Cooperativity
association constant vs. dissociation constant for bimolecular interaction
equation relating fractional binding to Kd - know how to interpret hyperbolic curve
experimental steps needed to determine Kd
using competition to assess affinity - meaning of IC50; how to determine IC50
cooperativity factor (alpha) for three component system (A,B,C)
experimental steps needed to determine cooperativity factor (alpha)
use of Hill equation and Hill plots - What is plotted? Hill coefficient, degree of cooperativity