FINAL EXAM STUDY GUIDE BIOE 201 TOPICS FROM OTHER EXAMS Sizes of cells molecules etc Eukaryotic cell ave diameter of 20 um E coli bacteria prokaryotic 1 um o Significantly smaller than your cells Protein diam 5 nm o 1 nm is 10 3 um H ion diameter 0 1 nm o 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 proteins Zero order A B o d A dt k units M s First order A B o d A dt k A units s 1 Second Order A B C Kd kon koff orders and units of rate constants o d C dt k A B units M 1 s 1 kon and koff are the rate constants kd is the dissociation constant o 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 associations In proteins between H and an open pair of electrons o Hydrogen Bonds o Ionic Bonds Attraction of opposite charges Electrons are not shared o 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 page o SDS negatively charged detergent Denatures protein Also boil sample to denature Makes everything negative Binds hydrophobic residues o Polyacrylamide crosslinked gel o Reducing agent reduces disulfide bonds o Separates size of protein Isoelectric focusing o Separates by charge pH dependent o COOH and NH2 groups o Stops at equilibrium point 2 D Gels o 2 dimensional electrophoresis o Separates by charge and mass o Detects protein finger print o Figure out protein using mass spec Antibodies o Uses Detection of specific proteins Protein and cell purification Separate specific proteins or cells from complex mixture Inhibitors Enzymes or cell adhesion molecules o Produced by B cells o Bind to unwanted proteins cells toxins ANTIGENS o Inactivate or tag for removal o Structure bifunctional Antigen binding domain Effector domain o Disulfide bonds w in and between chains o Monoclonal antibodies are used to isolate single species from immortal cells Column chromatography o Most common way to isolate proteins o Separates by physical characteristics o Purification of protein from complex mixture o Types Ion exchange Separation by charge Separation by size Gel filtration 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 density o 2 DNA spotting Synthesize 20mer primers or cDNA in aqueous form Prepare membrane or glass Spot primers to desired locations Ways pipet pen inkjet printer o high sensity arrays very miniaturized want redundancy mult Primers gene o 3 Labeling 10 6 spots chips Use two diff fluorescent dyes Label in parallel reactions hybridize together Can incorporate fluorescent bases into cDNA o Not good for absolute determination of concentrations o Not good for minute samples o Genes turned on to different degrees o What groups of genes are co regulated o Expression profiles of unknown genes o Diagnostics about diseased tissue Ligand binding and enzyme M M kinetics Fractional occupancy assumptions o Rtot number of unbound receptors number of bound receptors o Big bath Ltot is approx ligands just in solution o If kon then high affinity to bind and reaction moves forward faster reaction LR L L kd Rtot Kd concentration of ligand when receptors are occupied Etot enzymes in complex free enzymes To increase productivity of enzyme anything to increase velocity o Increase substrate affinity o Decrease km o Increase enzyme amount o Not ligand or substrate amount those depend on where in the reaction you increase them o Increase in velocity is a great way to decrease km Km 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 proteins stop codon Plasmid structure o Enhancer Promoter ribosome binding site start codon ORF sequence Transcription creating mRNA from DNA o Transcribed DNA is stitched back together o Reads ANTICODING strand to create replica of CODING 5 3 reads 3 to 5 o recognizes promoter first o synthesis begins start codon introns non coding reagions exons coding regions splicing creates meaningful sequence o alternative splicing creates different proteins from the same gene caps 5 end polyadenylation on 3 end o 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 energy o Trp activates repressor When absent polymerase allowed to bind genes on When present binds to repressor turns genes off o Negative feedback Lac operon o Don t want it on if glucose is present and lactose is absent o Wants NO glucose YES lactose o 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 regulation Repressor binds to promoter in absence of lactose Lactose inactivates repressor Expression and purification of recombinant proteins in bacteria I have a hypothesis that a certain growth factor receptor x we ll call it GFRx becomes activated in brain tumors and I want to study this receptor to understand it better Provide steps for the following protocols Each recipe should be on the order of 10 steps or so 1