FINAL EXAM STUDY GUIDE BIOE 201TOPICS FROM OTHER EXAMS:Sizes of cells, molecules, etc.- Eukaryotic cell – ave diameter of ~20 um- E. coli bacteria (prokaryotic)- ~ 1 umo Significantly smaller than your cells- Protein – diam ~5 nmo 1 nm is 10^-3 um- H+ ion – diameter ~0.1 nmo Also length of a C-C bond- Cell membrane - ~ 3 nm thick- 1 angstrom = 0.1 nm = 10^-10 m- Alpha helix 0.54 nm- 3.6 amino acids/turn (DNA)- Protein = 50 kDa- Weight of amino acid: 110 Da- 450 amino acids/ proteinsKd, kon, koff, orders and units of rate constants- Zero order – A  B o d[A]/dt = k units: M/s- First order – A  Bo d[A]/dt = k[A] units: s^-1- Second Order – A + B  Co d[C]/dt = k[A][B] units: M^-1 s^-1- kon and koff are the rate constants, kd is the dissociation constanto kD = koff/kon (important for michaelis menten)- keq = [prod]/[reac] = exp(-deltaG/RT)Intermolecular forces (noncovalent)- energy required to break noncovalent interactions is only slightly greater than average KE at room temp- important in protein folding and intermolecular associationso Hydrogen Bonds In proteins, between H and an open pair of electronso Ionic Bonds Attraction of opposite charges Electrons are not sharedo Van der waals interactions A weak, nonspecific attractive force created due to momentary random fluctuations that produce a transient electric dipole - Non polar molecules adhere because of hydrophobic forces- Multiple noncovalent bonds can confer binding specificity- Relative strengths: (weak) van der waals, hydrogen, carbon, ionic (strong)Methods of detecting proteins, DNA, RNA- X-ray crystallography: determine overall protein structure- SDS-pageo SDS – negatively charged detergent Denatures protein- Also boil sample to denature Makes everything negative Binds hydrophobic residueso Polyacrylamide – crosslinked gelo Reducing agent reduces disulfide bonds o Separates size of protein- Isoelectric focusingo Separates by charge (pH dependent)o COOH and NH2 groupso Stops at equilibrium point- 2-D Gelso 2 dimensional electrophoresiso Separates by charge and masso Detects protein “finger print”o Figure out protein using mass spec- Antibodieso Uses: Detection of specific proteins Protein and cell purification- Separate specific proteins or cells from complex mixture Inhibitors- Enzymes or cell adhesion moleculeso Produced by B-cellso Bind to unwanted proteins, cells, toxins (ANTIGENS)o Inactivate or tag for removalo Structure: bifunctional Antigen binding domain Effector domaino Disulfide bonds w/in and between chainso Monoclonal antibodies are used to isolate single species from immortal cells- Column chromatographyo Most common way to isolate proteinso Separates by physical characteristicso Purification of protein from complex mixtureo Types: Ion-exchange- Separation by charge Gel-filtration- Separation by size Affinity- Substrate for enzyme, antibody, DNA oligonucleotide, Ni2+, DNA or proteins- Separation by specific binding to another molecule- Northern Blot – detects mRNA- Western Blot – detects specific proteins/ amino acids using antibodies- Southern Blot – using gel electrophoresis to separate DNA fragments, detects specific DNA sequence- DNA Microarray:o 1. Immobilize DNA (know sequence looking for) A. make prime for specific sequence B. use cDNA for gene of interest C. synthesize primers on chip- Most automated- Mass production- Highest densityo 2. DNA spotting Synthesize 20mer primers or cDNA in aqueous form Prepare membrane or glass Spot primers to desired locations- Ways: pipet, pen, inkjet printero high sensity arrays very miniaturized want redundancy (mult. Primers/ gene) 10^6 spots/chipso 3. Labeling Use two diff. fluorescent dyes Label in parallel reactions, hybridize together Can incorporate fluorescent bases into cDNAo Not good for absolute determination of concentrationso Not good for minute sampleso Genes turned on to different degreeso What groups of genes are co-regulatedo Expression profiles of unknown geneso Diagnostics about diseased tissueLigand binding and enzyme (M-M) kinetics- Fractional occupancy assumptions:o Rtot  number of unbound receptors + number of bound receptorso Big bath: Ltot is approx. ligands just in solutiono If kon >> then high affinity to bind and reaction moves forward, faster reactionLR = (L/(L +kd))*Rtot- Kd: concentration of ligand when ½ receptors are occupied- Etot: enzymes in complex + free enzymes- To increase productivity of enzyme: (anything to increase velocity)o Increase substrate affinityo Decrease kmo Increase enzyme amounto Not ligand or substrate amount, those depend on where in the reaction you increase themo Increase in velocity is a great way to decrease kmKm = [E][S]/[ES] V= Vmax([S]/(km+[S])) Vmax = Etot(kcat) dP/dt = kcat[ES]Graph of Vmax vs. [S] Gene structure and regulation of transcription- Genes code proteinso Enhancer  Promoter  ribosome binding site  start codon  ORF (sequence)  stop codon- Plasmid structure- Transcription – creating mRNA from DNAo Transcribed DNA is stitched back togethero Reads ANTICODING strand to create replica of CODING (5’ – 3’) (reads 3’ to 5’)o recognizes promoter firsto synthesis begins @ start codon- introns – non coding reagions- exons – coding regions- splicing creates meaningful sequenceo alternative splicing creates different proteins from the same gene- caps 5’ end, polyadenylation on 3’ endo prevents bonding, IDs as RNA (only in eukaryotes)- transcription factors: protein that binds to DNA and alters transcription levels of specific genes- activators – turn up transcription- repressors – turn down transcription- promoter- DNA sequence upstream of gene where RNA polymerase binds- operon – group of genes that are co-regulated- Trp operon:o Wants to synthesize trp when absent, when trp is present, wont waste energyo Trp activates repressor When absent, polymerase allowed to bind, genes on When present, binds to repressor, turns genes offo Negative feedback- Lac operono Don’t want it on if glucose is present and lactose is absento Wants: NO glucose YES lactoseo Use pos. and neg. regulation to gene expression A. Positive regulation- CAP activates transcription- Polymerase binds weakly to CAP and DNA (lac control region)o Comb. of free energies is enough to make it bind and transcribe- NOT glucose- CAP bends DNA around itself B. Negative