Biochemistry Exam 2

Protein Source

Proteins can be extracted from the cells of any organism known to express it Pros: -Native environment- more likely to have appropriate PTM, fold correctly, and be around the correct cofactors Cons: -The amount of that particular protein might be quite small, and could be challenging to scale up -Extracting your protein from a particular source makes modifying the amino acid sequence impossible-restricts experimental possibilities and makes purification more challenging -A better way to do this is to use a clone factor with that particular mutation It is now far more common to express the protein in culture from a model organism:E. coli, yeast, insect cells mammalian cells -Allows for overexpression and easy scale up -Different model organisms are used for the protein source -Sex cells from mammals-you don't get nearly as much protein as in bacteria and yeast -Grow plasmid in a small flask and then naturally expand this -Culture cells by growing them up in a rich media, turn on gene expression, then isolate protein

Protein Evolution

Proteome-Amino acid sequences of all of the proteins encoded by the genome -Random mutational events that alter the protein structure lead to evolution -Sequence differences in DNA and proteins can be used to construct evolutionary trees -Sequences retain a record of evolutionary history -Evolution recycles genes into new functions -Many mutations are deleterious and often lethal in their effects and therefore rapidly die out Mutations are propagated only if: -They increase the likelihood that the owner will survive to reproduction otherwise a mutation will be lost - They do not change this likelihood, or have a mild change-these will accumulate over time and alter evolutionary trajectory

Organizing the Complete Primary Structure

Span the Cleavage Points and Arrange in Order: -With the peptide fragments individually sequenced, what remains is to elucidate the order in which they are connected in the original polypeptide -Do so by comparing the amino acid sequences of one set of peptide fragments with those of a second set whose specific cleavage sites overlap those of the first set -The overlapping peptide segments must be of sufficient length to identify each cleavage site uniquely -As there are 20 possibilities for each amino acid residue, an overlap of only a few residues is usually enough Determine the Positions of the Disulfide Bonds: -The final step in an amino acid sequence analysis is to determine the positions (if any) of the disulfide bonds -This is done by cleaving a sample of the native protein under conditions that leave its disulfide bonds intact -The resulting peptide fragments are then separated by reverse-phase HPLC(Dw about this) -Disulfide-linked peptide fragments are easily identified because, for each such linkage, two of the original peptide fragments will be replaced by a larger one -The disulfide-linked fragments are then sub- jected to Edman degradation -Although such a fragment yields two retention signals of amino acids in each step of this process (at least initially), their locations within the predetermined amino acid sequence of the protein are readily known, thereby establishing the positions of the disulfide bonds

Relative Elution Volume

Ve/Vo-A ratio designed to quantitatively analyze the outcomes of gel filtration chromatography-when this method is used as an analytical method -THe elution volume(Ve) is the volume of solvent required to elute the solute from the column after it has first contacted the gel -The void volume(Vo) of a column is easily measured as the elution volume of a solute whose molecular mass is larger than the exclusion limit of the gel. -The behavior of a particular solute on a given gel is therefore characterized by this ratio -This is independent of the size of the particular column used -Relation between this and the log of the molecular mass of various proteins for a particular column is used to estimate the molecular mass of an unknown protein -The precision of this technique is limited by the accuracy of the underlying assumption that the known and unknown macromolecules have identical shapes

Insulin

- 51-residue primary structure that was determined by Fredrick Sanger from bovine species -In mature form, consists of two AA chains linked by two intermolecular disulfide bonds -An intramolecular disulfide is also present. -The A chain has 21 AAs and the intramolecular disulfide -The B chain has 30 AAs -Strategy of Sanger: break the disulfides and then sequence each chain

Protein Separation for Sequencing

- Following disulfide cleavage, A protein's nonidentical polypeptides must be separated and purified in preparation for their amino acid sequence determination -Subunit dissociation and denaturation occur under acidic or basic conditions, low salt concentrations, elevated temperatures, or through the use of denaturing agents such as urea, guanidinium ion, or detergents such as sodium dodecyl sulfate (SDS) -The dissociated subunits can then be separated by purification methods capitalizing on small differences in polypeptide size and polarity -Ion exchange and gel filtration chromatography are most often used -It also is useful to know the number of residues- find molecular mass with SDS-PAGE or mass spec to be more accurate

Species vs Gene Tree

-A gene tree describes the evolutionary history of a particular gene, regardless of what organism has been its host -A species tree describes the overall evolutionary history among organisms using, in principle using the entire genome to determine relationships -To simplify things for constructing a species tree, 16S rRNA is a single gene that is often used as a proxy-RNA sequence to compare between two different organisms and find out how different they are based only on that sequence

Differential Centrifugation

-A method of protein purification-used in the step when you are clearing the cell lysate -A process in which the cell lysate is spun at a speed that removes only the cell components denser than the desired component -If the protein is soluble, the protein can be separated from the other cellular debris -Can be used to isolate the component of the cell that contains the protein from the others (i.e. a mitochondrial protein) -Used to separate organelles from debris as well -Used as an analytical technique-the speed of the centrifuge can tell you more about the structure of the protein which are isolating Mass-based separation via this process -Svedberg Units(S) represent the rate which particles will sediment -Smaller molecules will pellet at higher speeds(smaller S) -Large molecules/cellular components will pellet at slower speeds(higher S) -Almost any cellular component will spin down at a high enough speed

Cytochrome C

-A nearly universal eukaryotic protein involved in the electro transport chain function -Since over a billion years ago, this protein has changed very little, -If taken from a eukaryotic organism, it will react with other proteins involved in the same process, but from highly unrelated organisms -the amino acid sequences of the cytochromes c from over 100 widely diverse eukaryotic species ranging in complexity from yeast to humans is shown -The various residues in the table have been colored according to their physical properties in order to illuminate the conservative character of the amino acid substitutions - A total of 38 of its 105 residues are invariant and most of the remaining residues are conservatively substituted -The clear biochemical role of certain residues makes it easy to surmise why they are invariant-His 18 and Met 80 form ligands to the Fe atom of cytochrome c; the substitution of any other residues in these positions inactivates the protein -those that have weak conservation or those that have similar but slightly different types of amino acids in the same position -Those that have no conservation or unconserved pairing are hypervariable(Ex: valine and thymine at position 28-Acidic and basic) -This technique is useful for trying to find out if differences in sequence confer different activities or functions

Ion Exchange Chromatography

-A protein purification method based on separation by size -Ions that are electrostatically bound to a matrix are reversibly replaced by ions in solution -Anion exchangers bear positively charged groups and reversibly bind anions that are negatively charged -Cation exchangers bear negatively charged groups and reversibly bind cations that are positively charged -Therefore, Polyanions and polycations bind to anion and cation exchangers, respectively -Protiens are polyelectrolytes (polyionic polymers) that bear both positive and negative charges-can bind to both cation and anion exchangers depending on their net charge -The affinity with which a particular polyelectrolyte binds to a given ion exchanger depends on the identities and concentrations of the other ions in solution -the competition among various ions in solution for the binding sites on the ion exchanger effect its affinity of the protein -Proteins bear acid-base groups, so their affinity to exchangers are also highly pH dependent because of the variation of their net charges with pH Process: -A small amount of impure protein is added to the top of a column in which the ion exchanger is packed -As the column is washed with a buffer, proteins with relatively low affinity proteins move through the column faster than ones with higher affinities -The progress of a given protein through the column is retarded relative to that of the solvent due to interactions between the protein and the ion exchanger -Proteins that bind tightly to the ion exchanger are eluted by changing the elution buffer to one with a higher salt concentration (and/or a different pH), a process called stepwise elution -With the use of a fraction collector, purification of a substance can be effected by selecting only those fractions of the column eluant that contain it -Detection of the separated substances using UV visible absorption spectra along different points in your purification

6-His Tag

-A tag inserted into a sequence of choice to be cloned encoding 6 sequential histidines -Unlikely to be naturally encoded by a protein -Resin is a Matrix of NTA bound to nickel or cobalt can chelate Histidine residues -Imizadole is used to elute of the His-tagged protein after washing

Silver Stain

-A visualizing agent like Coomassie Blue used after gel electrophoresis -~50x more sensitive than Coomassie Blue -Only tells you that there is protein of the approximate size you predict and its relative purity, it does not confirm identity

Protein Purification Overview

-After isolating your gene and putting it into an appropriate plasmid and enrich under a proper promoter -Instead of being 1% of your cellular mass, under proper promoter it can be made the primary component in a bacteria cell -Then you lyse your cells and add this mixture to some sort of column or purification matrix that will bind your protein of interest and wash off contaminants and other protein(fractionating) -there are all sorts of fractionating methods-size charge etc.

Protein Fragmentation for sequencing

-After preparation, separated subunits are fragmented depending on size -Polypeptides that are longer than 40 to 100 residues cannot be directly sequenced -Polypeptides of greater length must be cleaved enzymatically or chemically to fragments small enough to be sequenced -Trypsin cleaves specifically after basic AAas like Arg and Lys if the next residue is not Pro-this lets you know that if you have a product probably lysine or arginine at the end-gives you a pattern of cleavage -At this point, it is still difficult to determine the specific sequence because using same kind of protease yields specific patterns of cleavage - this would not allow you to have overlaps in sequencing -Cleave another sample of the protein using cyanogen bromide (cleaves after Met) -Both proteases have a reproducible pattern of fragmentation-obtain overlapping fragments for sequencing

Gel filtration Chromatography

-Aka size exclusion chromatography -Molecules are separated by size (primarily) but shape can also play a role -Beads containing pores of a given size act as molecular sieves -Proteins larger than the pore bypass the pore and transit quickly -Proteins smaller than the pore enter the pore, slowing their transit -The gel's exclusion limit refers to the size of the smallest molecule unable to pass through the pores -Elongated molecules will behave like larger molecules as compared to more spherical molecules-Most proteins are globular though -Molecules with molecular masses ranging below the exclusion limit of a gel will separate in the gel in the order of their molecular masses, with the largest eluting first -This is because pore sizes in any gel vary over a limited range-larger molecules have less of the gel's interior volume available to them than do smaller molecules -Size and elution are linearly related, so this can be used to estimate molecular weight -Take a mixture of molecules of known molecular weight and run them over the column, get the right number of peaks -Plot their relative elution volume versus molecular weight when they come off the peak-get a linear curve -THEN run your sample, plot your sample's relative elution volume and find the mass -Also can use this technique to find out if proteins from complexes, such as a dimers-you would do your elution and see that your protein comes off at a higher volume then you would predict the protein has formed some sort of complex rather than behaving as a monomeric protein

Western Blot

-Also known as Western Blot -Avariation of Southern blotting that uses a technique similar to ELISA to detect protein(s) of interest - Used ifstong validation is needed that a purified protein is the protein that you are looking for, and not just the right molecular weight(limitation od Coomassie/Silver Stain) Process 1. A completed gel electrophoretogram is blotted onto a sheet of nitrocellulose(a transfer film), which strongly and nonspecifically binds proteins [nylon or polyvinylidene fluoride (PVDF) membranes may also be used]. 2. The excess adsorption sites on the nitrocellulose are blocked with milk because it had a lot of proteins with a lot of different molecular weights to prevent the nonspecific adsorption of the antibodies (which are proteins themselves) used in Steps 3 and 4. 3. The blot is treated with antibody to the protein of interest (PRIMARY ANTIBODY). This is usually from a rabbit. None of the proteins in milk will bind antibody, so the antibody should bind only the specific sites on the gel that contain the protein of interest ***Alternatively, the primary antibody may be labeled with the radioactive isotope 125I and the position of the bound protein on the blot revealed by autoradiography after washing away unbound antibody 4. After washing away the unbound primary antibody, the blot is incubated with a SECONDARY ANTIBODY(from a goat or other non-rabbit species) directed against all rabbit antibodies, to which an easily assayed enzyme(such as a fluorophore) has been covalently linked ***An advantage to using a secondary antibody is that some primary antibodies have multiple recognition sites-therefore you have multiple secondary antibodies per primary antibody leading to single application which makes it easier for detection of smaller samples 5. After washing away the unbound secondary anti- body, the enzyme in the bound secondary antibody is assayed with a color-producing reaction, causing colored bands to appear on the nitrocellulose where the protein of interest is bound.

Native Chemical Ligation

-Alternative peptide synthesis technique to SPPS for intermediate length peptides -Much cheaper than SPPS -Two peptides can be linked to make peptides up to 120 residues long -Sequentially, you can do this to yield proteins as big as 203 aa

Salting out

-As Ionic strength increases, the solubilities of proteins as well as other molecules increases -Result of the competition between the added salt ions and the other dissolved solutes for molecules of solvation -At high salt concentrations, so many of the added ions are solvated that the amount of bulk solvent available becomes insufficient to dissolve other solutes. -The downhill fall of the ionic strength vs solubility curve -often an early step in purification-gradually dumping salt into protein mixture and measuring at what concentration the protein precipitates out

Clustal

-BLAST makes only pairwise alignments -This simultaneously aligns more than two sequences(peptides of DNA-Multiple sequence alignment tool -Similarity scores are calculated for all the different residues for similarity -Also weighs overall conservation of mutations -Confidently aligned residues are shaded according to residue type -The bar graph below the alignment is indicative of the alignment quality -Useful If you want to compare certain known sequences to each other

BLAST

-Basic Local Alignment Search Tool -Pairwise sequence alignment software -A query sequence is entered and it returns a list of sequences ( in the genome or proteome)that are most closely similar -Protein that is never been discovered and want to figure out what it does-Use this tool to find out what kind of protein it is From the figure Green-identify the subject sequence and indicates its length in residues Black-Assortment of alignment statistics Blue-The query and subject sequences are then shown vertically aligned with the line between them (black) indicating residues that are identical (by their one-letter codes) and similar [by a plus (+)] -The output of BLAST consists of a series of such pairwise alignments

Homologs

-Both Paralogs and arthologs are these -This just means that the genes have shared ancestry

Molecular Clock

-By analyzing the differences of enzymes from major groups, this can provide reliable estimates of when these groupings diverged. -Based on the supposition that homologous sequences diverge at a uniform rate, which was calibrated using sequences from vertebrates for which there is a reliable fossil record -Suggests that animals, plants, and fungi last had a common ancestor ~1 billion years ago -What drives evolution? Likely not only protein sequences -Human and chimpanzee genomes are 99% identical, and proteins typically have only 2aa differences each-Yet the anatomical and behavioral differences between human and chimpanzee are so great that these species have been classified in separate families -This suggests that the rapid divergence of human and chimpanzee stems from relatively few mutational changes in the segments of DNA that control gene EXPRESSION-how much of each protein will be made, where, and when

Nucleic Acid versus Peptide Sequencing

-By the late 1970s, DNA sequencing methods had advanced to the point that it became far easier to sequence a DNA segment than the protein it specified -A majority of known protein primary structures have been inferred from DNA sequences -Direct protein sequencing remains an important biochemical tool for several reasons: 1. Disulfide bonds can be located only by protein sequencing. 2. PTM can be determined only by directly sequencing the protein. 3. To identify the gene encoding a protein of interest determine the amino acid sequence of at least a portion of the protein-Reverse genetics -Using this, one can infer the base sequence of the DNA segment that encodes this polypeptide segment, chemically synthesize this DNA, and use it to identify and isolate the gene containing its base sequence through Southern blotting or PCR

Solubilizing the Protein

-Cells pelleted from growth phase must be broken open to access the protein -Animal cells can typically be lysed by osmosis in a hypotonic solution: water diffuses into the cell and the cells burst -Bacteria, plants, and yeast have a cell wall and require harsher lysis methods How to conduct cell disruption -Lysozyme is typically used to degrade the cell wall along with mechanical disruption: -Uhhhh use aa mf blender -Sonicator-uses sonic waves to open the cell -French pressure cell-most complex-feed cells through a component and subject them to high pressures-most gentle on the protein, but harshest on cell -Once you have broken open cells with cellular debris-do not want to pass through column yet-columns are expensive and debris will clog them

Protein Solubility

-Certain proteins precipitate from solution under conditions in which others remain quite soluble. -At a given ionic strength this varies with the types of ions in solution and the concentration(Ionic strength) -Some ions can denature proteins, and do not salt them out -Salt generally increases the solubility of protein at low ionic strength-can also be backwards but it depends of the protein which are considering -The order of effectiveness of these various ions in influencing protein solubility is similar for different proteins-mainly due to the ions' size and hydration -As salt increases the ions shield the protein molecules' charge(at low ionic strength), thus increase the protein's solubility (salting in)-Uphill climb of the curve on the right -As the salt becomes very high, the solubility decreases due to the competition of the salt and protein for solvation (salting out)-Downhill fall of the curve on the right How it is used -By adjusting the salt concentration in a solution containing a mixture of proteins to just below the precipitation point of the protein to be purified, many unwanted proteins are eliminated from the solution -After the precipitate is removed by filtration or centrifugation, the salt concentration of the remaining solution is increased to precipitate the desired protein

Trypsin

-Degestive enzyme with the greatest specificity out of the peptidases used for protein sequencing -Cleaves peptide bonds on the C-side (toward the carboxyl terminus) of the positively charged residues Arg and Lys if the next residue is not Pro -Other peptidases can be used in limited proteolysis reactions to fragment at a lower level

ESI-MS

-ESI-based mass spectrum -Gas phase macromolecular ions are directed into the mass spectrometer, which measures their m/z values with an accuracy of 0.01% -If an ion's z value can be determined, its molecular mass can be determined with far greater accuracy than by any other method -The measured m/z ratios and the inferred charges for most of the peaks are indicated -Since most mass spectrometers are limited to detecting ions with m/z values less than several thousand, the use of ESI-MS has the advantage that the high ionic charges of the ions it produces has permitted the analysis of compounds with molecular masses 100 kD -Still doesn't really give you anything besides the mass

Denaturants

-Elevated temperatures -Urea -Guanadinium chloride(GuCl) -SDS

Limited Proteolysis

-Endopeptidases besides trypsinexhibit broader side chain specificities and often yield a series of peptide fragments with overlapping sequences -By adjusting reaction conditions and limiting reaction times, these less specific endopeptidases can yield useful peptide fragments -This is because the native structure of a protein buries many enzymatically susceptible peptide bonds beneath the surface of the protein molecule -Only those peptide bonds in the native protein that are initially accessible to the peptidase will be hydrolyzed -Limited proteolysis is often employed to generate peptide fragments of useful size from subunits that have too many or too few Arg and Lys residues to do so with trypsin -Although if too many are present, limited proteolysis with trypsin may also yield useful fragments

ELIZA

-Enzyme-liked immunosorbent assay -Used to detect small amounts of specific proteins and other biological substances in both laboratory and clinical applications -If a researcher just wants to know if a protein is in the cell, this is all you have to do after lysing the cell 1. An antibody against the protein of interest is immobilized on an inert solid such as polystyrene. 2. The solution being assayed for the protein is applied to the antibody-coated surface under conditions in which the antibody binds the protein. The unbound protein is then washed away. 3. The resulting protein-antibody complex is further reacted with a second protein-specific antibody to which an easily assayed enzyme has been covalently linked. 4. After washing away any unbound antibody-linked enzyme, the enzyme in the immobilized antibody-protein-antibody-enzyme complex is assayed, thereby indicating the amount of the protein present

Protein Functions

-Enzymes that catalyze reactions -Hormones and receptors for hormones that transmit messages -Transport biologically important substances like metals, oxygen, glucose, and lipids.

Example Workflow:SDS-PAGE

-Following Affinity Chromatography, you want to assess the purity of your sample to see if you need to conduct further chromatography -Run an SDS page gel assessing the Ni Eluate column of the gel -Pre-induction means before you induced the expression of the protein-a lot of other proteins in the cell -Lysate means after you induce and lyse the cells-shows nice enrichment of the band of the protein that you overexpressed-the protein of interest, Hsp104(seen at 104 kDa-good) -The load is what is actually loaded into the nickel purification column -FT(flow-through)-this is everything eluted off of the protein-lost a little bit of your protein(O.K.) -After you elute the column, the protein of interest is relatively pure with faint shows of contamination -For further purification, proceed to anion exchange chromatography

Example Workflow: Size Exclusion Chromatography

-Following a second chromatography procedure, If you want to purify even further, say for crystallization, perform size exclusion chromatography -this'll help to exchange the protein and get it into a appropriate final buffer and get rid of any remaining contaminants -No other significant bands can be observed in the gel -At this point you are ready to do high purification demanding techniques such as crystallography

The Peptide Bond

-Has a rigid, planar structure -This is a consequence of resonance interactions that give the peptide bond an 40% double-bond character

Conservatively Substituted Residues

-Have less stringent side chain requirements so that only residues with similar characteristics (e.g., those with acidic properties: Asp and Glu) are required

Ion exchange chromatography using stepwise elution

-Here the tan region of the column represents the ion exchanger and the colored bands represent the various proteins (a) The protein mixture is bound to the topmost portion of the ion exchanger in the chromatography column (b) As the elution progresses, the various proteins separate into discrete bands as a consequence of their different affinities for the ion exchanger under the solution conditions -The first band of protein (red) has passed through the column and is being isolated as a separate fraction, whereas the other, less mobile bands remain near the top of the column (c) The salt concentration in the elution buffer is increased to increase the mobility of and thus elute the remaining bands (d) The elution diagram of the protein mixture from the column

Orthologs

-Homologous proteins/genes in different organisms that arose through species divergence -various cytochromes can be used as an example

Ion Strength

-How the salt concentration is represented in salting in and salting out curves -Depends on the concentration of the salt in the solvent and the ionic charge of the species

Example Workflow: Anion exchange Chromatography

-If the initial affinity chromatography did not work, anion exchange chromatography can be used to further rid of impurities -Starting at low-salt, you increase the salt concentration creating a gradient -Obtain a junk peak with no real shouldering corresponding to our protein -Isolate and run different elution volumes on the second peak through a gel to see if there are any other proteins that appear in the gel with the protein -If you see another band you know that this elution should be not used

Protein Separation Methods

-If the properties of the protein are somewhat understood, these can be used in the purification -How do you know the properties if it is a new protein-Often trial and error -Qualities that the protein that might have that would enable them to be separated -Salting in and salting out or the two original techniques-using a certain salt to get the protein to precipitate out -Polarity separations are more use for peptides and proteins-most people use ionic charge or molecular size for separation -Most popular technique now is affinity chromatography

Factors of Protein Purification

-In the cell, the protein has the correct conditions- cofactors, solute, and compartment -When you break open the cell also to problems arise-temperature fluctuations, proteases, stickiness of protein Important things to do during purification: 1. Keep things as quick as possible both with purification and assays 2. Keep everything cold-at 4ºC 4. Be aware of proteases(enzymes that catalyze the cleavage of peptide bonds-abundant in cells)-Use protease-deficient strains, protease inhibitors, and freeze immediately after doing assays-proteases work well at higher temperatures 3. Minimize adsorption/aggregation-keep the protein concentrated, use small amounts of detergent, and avoid dialysis and filtration if possible 4. Be a good accountant-Use quantitative assays to keep track of yield, purity, and activity at each step -Until a protocol is optimized, trial and error with careful analysis is essential-you don't really know how proteins will behave in salts and solutes until you try

E. Coli Expression Workflow

-Inoculate a small starter culture and grow in culture -Get the culture to the point where the cells can express protein the best-this is when the cells are growing healthily and rapidly -Turn on gene expression using IPTG or other type of inducers for appropriate promoters -Collect your cells at the end of the day in a centrifuge - Freeze many small amounts of cells in pellets for further purification

Secondary Structure

-Local spatial arrangement of a polypeptide's or a nucleic acid's backbone atoms without regard to the conformations of their side chains or bases.

Protein Assay

-Means to detect the presence of a protein-critical for protein purification -Proteins are typically in solution, and the solution should be clear with no precipitate (this means that your protein is inactive-insoluble)-a method for detecting the active protein/successful purification is essential -Enzymes catalyze reactions, so their activity can be measured directly -Spectroscopic techniques can be used to detect if the proteins there-doesn't tell you anything about specific activity of the protein, however -Binding to a known target can be used to detect non-enzymatic proteins

Trypsin Fingerprinting

-Method of fingerprinting -Unknown protein is cut out of an SDS-PAGE gel -Protein is digested using trypsin -Then the fragments are massed using MALDI-TOF or ESI-TOF -Masses are compared to a database of known protein sequence digests

Chromatography

-Mixture of substances to be fractionated is dissolved in a liquid or gaseous fluid known as the mobile phase -The resultant solution is percolated through a column consisting of a porous solid matrix known as the stationary phase, which in certain types of chromatography may be associated with a bound liquid -The interactions of the individual solutes with the stationary phase act to retard their progress through the matrix in a manner that varies with the properties of each solute -If the mixture being fractionated starts its journey through the column in a narrow band, forces on each component that cause them to migrate at different rates will eventually cause the mixture to separate into bands of pure substances -More preferable than salting out -Often several columns will be used to sequentially purify a given protein (i.e. first charge, then size) -Tags (6-His, GST, MBP) are frequently employed as an initial enrichment chromatography step-you can pull down anything that has this tag in an early step Types: -Affinity chromatography: relies on a specific tag -Ion exchange chromatography: relies on charge -Size exclusion chromatography: relies on size/shape -All rely on a stationary phase (resin-beads) and a mobile phase (protein solution)

Coomassie Blue

-Most most common visualizing agent for detecting ABUNDANT proteins after gel electrophoresis -Detects 100ng-30ug protein -Signal is proportional to protein amount -Only tells you that there is protein of the approximate size you predict and its relative purity, it does not confirm identity From the Figure: -The two wells on the left are the impure cell lysate-Notice that the protein of interest is overexpressed, as expected -This confirms that the protein in the gel by itself is the protein of interest -From the indicated gel, you can see that the protein still not pure enough unless it is an early step

Nucleic Acid Purification

-Most of the methods of protein purification, often with some modification, are also regularly used to fractionate nucleic acids according to size, composition, and sequence- some are applicable only to nucleic acids -Nucleic acids in cells are invariably associated with proteins- their nucleic acids must be deproteinized when seeking to isolate from a cell ISOLATION: -Shaking an aqueous solution containing the protein-nucleic acid complex with a 25:24:1 mixture of phenol, chloroform, and isoamyl alcohol -The protein is thereby denatured and extracted into the organic phase, which is separated from the nucleic acid-containing aqueous phase by centrifugation SEPARATION: -Polyacrylamide gel electrophoresis can be only used for small DNA fragments -DNAs of more than a few thousand base pairs are too large to penetrate a polyacrylamide gel -This is partially overcome through the use of agarose gels-relatively large DNAs in various size ranges may be fractionated- plasmids, for example, may be separated from the larger chromosomal DNA of bacteria DETECTION: -Gels are stained with an intercalating agent-often ethidium bromide- essentially this interpolates into the DNA between base pairs -UV light allows for visualization of the ethidium bound DNA

Quaternary Structure

-Most proteins are composed of two or more polypeptide chains, loosely referred to as subunits, which associate through noncovalent interactions and, in some cases, disulfide bonds -This level of protein structure refers to the spatial arrangement of its subunits

Gene Duplication

-Most proteins have extensive sequence similarities with other proteins from the same organism(paralogs) -Such proteins arose through this -Result of an aberrant genetic recombination event in which a single chromosome acquired both copies of the primordial gene in question -Particularly efficient mode of evolution-one of the duplicated genes can evolve a new functionality through natural selection while its counterpart continues to direct the synthesis of the presumably essential ancestral protein

Protein Sequencing

-Not done very often -Only do when concerning post-translational modifications -Or if a protein is unknown and want to know what DNA sequence it came from 2 METHODS 1. Edman degradation (classical method) -Successive rounds of N-terminal modification, cleavage, and identification -Can be used to identify protein sequence from N to C-terminus through iterative rounds of cleavage and sequencing -Useful for up to about 50 amino acids-beyond that it is impossible to get a good signal 2. Mass spectrometry (modern method) -MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence -Can also be used to determine posttranslational modifications - Edman degradation is primarily used to figure out the N terminus-other than that it's not very frequently used -Pretty much everything now is done using mass spec

Chromatography

-Often used in protein purification-Example workflow shown -Using a property of the protein to get it to stick to a resin-as selective as possible so just the protein of interest sticks to a resin -When done properly, just have your protein bound to the resin-in a frit -Beads are washed in a column over many cycles to get rid of any other proteins or components -Last step is eluting the protein so that you only have the protein hopefully in your final solution - There are many different types of columns and purification techniques-researchers often use these in combination to get certain component that you want in high-purity

Crystallization

-Once a protein has been brought to a reasonable state of purity, this is possible-Protein needs to be very pure -The protein solution is brought just past saturation point -After some time (hours to years), while slowly adding a precipitating agent, the protein may precipitate in crystalline form -It may be necessary to attempt the crystallization under different solution conditions and with various precipitating agents before crystals are obtained -The structure can be solved using x-ray crystallography once crystals are obtained

Edman degradation

-Once the manageably sized peptide fragments that were formed through specific cleavage reactions have been purified and separated, their amino acid sequences can be determined -This is done through repeated cycles of the Edman degradation, the classic method of amino acid sequencing -Consists of N-terminal modification, cleavage, and identification -Can be used to identify protein sequence from N to C- terminus through iterative rounds of cleavage and sequencing-up to about 50 amino acids-beyond that it is impossible to get a good signal -This is usually done with multiple sets of fragments, in order to obtain overlapping sequences to bemused in the nexts steps of organizing the complete structure Process: -Label and then cleave one amino acid at a time from the N-terminus of a protein -The cleaved AA is passed through an HPLC and identified based on known standards-the retention time of the signal are able to compute where and when the amino acid was taken often at what position -Up to 50 AAs can be determined reliably using the technique -Beyond 50 AAs, the signal is typically lost

Fingerprinting

-Once the primary structure of a protein has been elucidated, that of a nearly identical protein, such as one arising from a closely related species, a mutation, or a chemical modification, can be more easily determined -This method combines MS with protease digestion - Protein is fragmented in a reproducible manner - Resulting peptides are separated to yield a specific pattern that can distinguish differences between related proteins -The peptide fragments incorporating the amino acid variations migrate to different positions on a gel than do the corresponding peptides of the original protein -Variant peptides could then be cut out of a gel, such as an SDS-PAGE gel and sequenced -Changes in the protein can be seen without the need to sequence it in its entirety-big advantage

Matrix-assisted laser desorption/ionization and Time-of-Flight(MALDI-TOF)

-One of the methods to vaporize and ionize macromolecular fragments in EM -A macromolecule is embedded in a crystalline matrix of a low molecular mass organic molecule (prepared by drying a droplet of solution containing the macromolecule and a large excess of the organic molecule) -Matrix is irradiated with intense short(nanosecond) pulses of laser light at a wavelength absorbed by the matrix material(not the macromolecule!!) -Considered a soft ionization" technique because energy from the laser is absorbed by the matrix but not the protein -Energy absorbed by the matrix ejects the intact macromolecules from its surface into the gas phase -Protein in the gas phase goes into a time-of-flight matrix unit, where the detector detects how fast the fragments get up to it -This gives you an indication of what the mass to charge ratio is with a spectrum -Doesn't really give you anything besides the mass

Electrospray Ionization(ESI)

-One of the methods to vaporize and ionize macromolecular fragments in EM -A solution of a macromolecule(analyte in a solvent) such as a peptide is sprayed from a narrow capillary tube maintained at high voltage ( 4000 V) -This forms fine, highly charged droplets from which the solvent rapidly evaporates -A series of gas phase macromolecular ions that typically have ionic charges in the range +0.5 to +2 per kilodalton -For polypeptides, the ionic charges result from the protonation of basic side chains such as Lys and Arg -Ends up giving a highly accurate mass of the protein-gives you a bunch of peaks of fragments with m/z versus relative abundances- add up the masses and get overall mass

Trans

-Peptide groups,with few exceptions, assume this conformation -Successive ⍺-C atoms are on opposite sides of the peptide bond joining them -This is partly a result of steric interference, which causes the cis conformation to be 8 kJ/mol less stable than this conformation -this energy difference is somewhat less in peptide bonds followed by a Pro residue-10% of the Pro residues in proteins follow a cis peptide bond

Salting In

-Phenomenon is that increases the solubility of a protein as the as the salt concentration of the protein solution increases -The additional counterions(of the salt) more effectively shield the protein molecules' multiple ionic charges and thereby increase the protein's solubility -The uphill climb of the ionic strength vs solubility curve

Disulfide Bond Cleavage

-Prior to this, N and C termini of the protein are determined to find out how many subunits are in the protein -This is done next in preparing a protein for protein sequencing -Often done reductively by treatment with sulfhydryl-containing compounds such as 2-mercaptoethanol of β-mercaptoethanol (BME) -The step after this is to separate and purify the sunbunits Done for 2 reasons: 1. To permit the separation of polypeptide chains (if they are disulfide linked). 2. To prevent the native protein conformation, which is stabilized by disulfide bonds, from obstructing the action of the proteolytic (protein-cleaving) agents used in primary structure determinations

Dialysis

-Process(less as a Purification and more as a exchange) that separates molecules according to size through the use of semipermeable membranes containing pores of less than macromolecular dimensions(kD) -These pores allow small molecules, such as those of solvents, salts, and small metabolites, to diffuse across the membrane but block the passage of larger molecules, such as proteins - A macromolecular solution is sealed inside a dialysis bag immersed in a relatively large volume of the new solvent -After several hours of stirring, the solutions will have equilibrated, but with the macromolecules remaining inside the dialysis bag -This process can be repeated several times to replace one solvent system completely by another Uses in purification -Very small protein contaminating your protein- perform dialysis sequentially diluting the contaminant away-replace buffer with new buffer every time -Following an ion exchange chromatography you finish with your protein in a high salt buffer-you don't want to leave your protein in it-put it your solution into a dialysis bag and allow the solution equilibrate with a buffer of lower concentration

Mass Spectroscopy

-Protein sequence determination is much easier with this method -Accurately measures the mass-to-charge (m/z) ratio for ions in the gas phase (where m is the ion's mass and z is its charge) -The method by which mass spectrometers produced gas phase ions is essential and difficult -often destroyed macromolecules upon vaporization by heating followed by ionization via bombardment with electrons -Two methods now used:Electrostray ionization(ESI) and Matrix-assisted laser desorption/ionization (MALDI) In the end, this technique gives us - MW and the molecular formula by % abundance -The molecules of a sample are bombarded with electrons, causing them to break apart and ionize -These ions are accelerated through a magnetic field and the resulting force deflects the ions around a curved path. -Radius of curvature of their path depends on the mass to charge ratio (m/z) of the ion -Most ions have a +1 charge -The magnetic field strength is altered to allow the passage of different sized ions through the flight tube and a computer records the amount of ions passing through at a given strength -Base peak: largest peak -"Parent peak": peak made by the molecular ions (original molecules that did not fragment, missing one electron -> +1) should be on the right side of the spectrum with heavy isotopes.

pH and Protein Solubility

-Protein side chains have a variety of pK's -For a given protein, there is an isoelectric point (pI): the specific pH where the positive charges balance the negative charges -At the pI, the protein carries no net charge, and thud it is immobile in an electric field -As the pH of the solution moves away from the pI, the protein's net charge increases and it is more soluble Important considerations: -If salting out is desired, salting out will be greatest if the pH is kept close to the pI -If a column purification in solution is employed, it is essential to keep the pH far away from the pI to avoid precipitation -Online calculators are available to calculate the pI from a specified amino acid sequence

Species Variation and Neutral Drift

-Proteins are constantly evolving -The present day sequences differ due to constant evolution, even once proteins are well adapted to their functions-even when sequences have evolved they still continue to pick up mutations -Comparisons of the primary structures of homologous proteins (evolutionarily related proteins) therefore indicate which of the proteins' residues are essential to its function, which are of lesser significance, and which have little specific function -You don't particularly converge on a proper sequence and then stop -Rather, once a protein has evolved into its present day function the likelihood of introducing a mutation that's more beneficial is much less than the likelihood of getting a detrimental mutation -Neutral drift- proteins will constantly acquire random mutations that change the protein sequence without altering protein function-often in conservatively substituted and hypervariable residues

Hypervariable Residues

-Residue position where many different amino acid residues may be tolerated -Indicates that the functional requirements of that position are rather nonspecific or confers some type of trait that organism might need for specific environment

Tandem Mass Spectrometry(MS/MS)

-Short polypeptides ( 25 residues) can be directly sequenced though the use of a tandem mass spectrometer-two mass spectrometers coupled in series -Repreat this process with every fragment: 1. The ion source generates gas phase peptide ions, P1, P2, etc. from a digest of the protein being analyzed. These peptides are separated by MS-1 according to their m/z values 2. One of them(P3 in the figure) is directed into the collision cell, where it collides with helium atoms, fragmenting it at specific sites 3. The fragments, F1, F2, etc. are directed into MS-2, where their m/z values are determined 4. Fragmentation Patterns are then assembled to yield the full sequence -By comparing the molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acid residues can be determined -Computerization of this comparison process has reduced the time required to sequence a (short) polypeptide to only a few minutes as compared to the 30 to 50 min required per cycle of Edman degradation -Highly reliable-computerized matching of a measured mass spectrum with those of peptides of known sequence as maintained in databases -Can be used to characterize post-translational modifications and locate disulfide bonds

Sickle Cell Evolution and Natural Selection

-Sickle-cell anemia patients have rythrocytes that form a sickle shape under low oxygen concentration, hindering their passage through blood cells, leading to severe problems -Sickle cell anemia was the first disease that was shown to arise from a single amino acid change in a protein -Pauling determined that normal hemoglobin had an anionic charge two units more negative than sickle cell hemoglobin -Ingram applied peptide fingerprinting to determine subtle differences in the peptides formed from tryptic digests -Sequencing has more recently shown that there is a Glu→Val mutation in the protein -Affects people throughout their lifetime-these types of mutations would not persist if they contain some problem prior to reproductive age-so why do we have the disease? -Individuals heterozygous for sickle-cell have hemoglobin that is ~40% sickled and still leave a normal life because their RBCs still have a long enough lifetime -Sickle-cell trait occurs mainly in those of equatorial African descent, where malaria is a major cause of death -Individuals heterozygous for sickle-cell trait are resistant to malaria

Chemical Synthesis of Polypeptides

-Smaller Polypeptides are synthesized by covalently coupling amino acids one at a time to a growing polypeptide chain attached to a bead-automated process -Synthesized from the C to N-terminus (reverse of translation) -Each amino acid being added must be chemically protected to avoid it reacting -This blocking group must be removed in order to add the next amino acid -N-terminus of oncoming amino acid and reactive side chains must be blocked during synthesis SPPS process -A growing polypeptide chain is covalently anchored by its C- terminus to an insoluble solid support such as beads of polystyrene resin -Appropriately blocked amino acids and reagents are added in the proper sequence -This permits the quantitative recovery and purification of intermediate products by simply filtering and washing the beads -When polypeptide chains are synthesized by amino acid addition to their N-terminus , the amino group of each sequentially added amino acid must be chemically protected during the coupling reaction -The tert-butyloxycarbonyl (Boc) group is frequently used for this purpose(Boc-Cl) -Drastically improves ease and yield -The peptide can easily be collected and washed due to it being anchored in place Limitations: -Limited to ~60 aa long peptides -Low yield -101 aa chain with 98% yield at each step, will give an overall yield of 2%-yield drops 2% every time -Many reactions may not go to completion and side products often form -Protecting groups may not be cleaved properly -As a result, must be purified by HPLC, further decreasing yield -End up losing quite a bit of peptide in the process -There are alternatives to this technique -If it's a large protein unit is express it in bacteria

SDS-PAGE

-Sodium dodecyl sulfate polyacrylamide gel electrophoresis -Sodium dodecyl sulfate(SDS) is a highly negatively charged detergent that masks the protein's intrinsic charge -Thus all proteins that travel in the gel are similarly charged -Typically proteins are dissolved in SDS containing sample buffer, boiled, and treated with reducing agent(β-mercapto-ethanol) to ensure the protein is denatured and disulfide bonds are cleaved -Proteins will migrate based on size only - SDS treatment disrupts the noncovalent interactions between these polypeptide subunits in proteins with multiple chains-this technique will yield the molecular masses of the protein's subunits rather than that of the intact protein -Typically proteins will run according to size-calibration curve of proteins run in a ladder can be made based on their relative migration versus their molecular weight -Mass and molecular motility have a linear relationship -Accuracy is usually +/- 10-15%

Allowed and Forbidden Polypeptide conformations

-Steric interference can occur between a carbonyl oxygen(red) and adjacent amide(blue) -By calculating the distances between the atoms of a peptide, allowed and forbidden Ф and ψ angles can be determined -From the different angles of the backbone one can predict where the sidechains are and how the different conformations of the backbone will be lead protein to form its structure -Any conformation where the interatomic distance is less than the corresponding van der Waals distance is forbidden -Atoms can be too far to close-too close and they repel each other-too far and they do not form stabilizing interaction -Ramachandran diagrams are used to summarize allowed and forbidden conformations

Ramachandran diagram

-Summarizes allowed and forbidden conformations based on torsional angles -77% of the Ramachandran diagram (most combinations of torsional angles) is conformationally inaccessible to a polypeptide chain. -With the known Van-der-Waal's radii limits, only three small regions of the conformational map are physically accessible to a polypeptide chain -However, all of the common types of regular secondary structures found in proteins fall within allowed regions of the diagram

Affinity Chromatography

-The ability of proteins to bind specific molecules tightly but noncovalently is harnessed to purify them by this method -A ligand that specifically binds to the protein of interest is covalently attached to an inert and porous matrix -Impure protein solution is passed through, and the desired protein binds to the immobilized ligand-other substances are washed through the column with the buffer -The desired protein can then be recovered in highly purified form by changing the elution conditions such that the protein is released from the chromatographic matrix -Protein tags are often used, Antibodies can be used but there are downfalls to using them: - Highly specific binding - Expensive - Binding is so tight, harsh elution is often necessary

Oligonucleotide synthesis

-The ability to chemically synthesize DNA oligonucleotides of specified base sequences is an indispensable part of cloning -Chemically synthesized oligonucleotides are commercially available and inexpensive (oligo 20 nt long costs ~$5) -New technologies are allowing for rapid synthesis of long oligonucleotides-now you can buy some there as long as 100 to 200 base pairs-you can link these to other oligonucleotides to create an gene -Degenerate bases can be incorporated to construct a library of sequences-where you want to observe whether a certain amino acid substitution will affect the overall function of the protein -Much more straightforward than native ligation-the process is a lot more easy in vitro than polypeptide synthesis - no one does this in their own lab-just send the sequence away and it's made for you

Proteomics

-The analysis of the protein content and properties of proteins in complex cellular environments -isolate a protein from anywhere in the cellular environment or you could take a whole organism -Digest with trypsin, put through MS, develop a profile of expression levels of different proteins in comparing samples -Analyze whole organisms, cells, or tissues to determine which proteins show increased or decreased expression profiles under different conditions -Won't yield the protein sequence, but rather how they change -Protein sequencing technology has allowed the rise of this

Torsional Angles

-The backbone of a protein is a linked sequence of rigid planar peptide groups -We can therefore specify a polypeptide's backbone conformation by the torsion angles of each of its amino acid residues - ⍺C-N - ɸ - ⍺C-C - Ѱ -Both of these angles are 180 in a fully -Side Chains have implications-angles can be restricted due to sterics -When viewed down the ⍺C, clockwise increases the angle, passing 180 makes the angle then negative

Phylogenetic Tree of Cytochrome C

-The easiest way to compare the evolutionary differences between two homologous proteins is simply to count the amino acid differences between them in various species and construct a tree based off the data achieved -These molecular trees generally agree well with trees based on other information such as morphology. -Indicates the ancestral relationships among the organisms which produced the proteins -Each branch point of a tree indicates the probable existence of a common ancestor for all the organisms above it -Nodes represent ancestral sequences-not necessarily present today but a common ancestor between species -The genes shown are orthologs-Homologous proteins/genes in different organisms that arose through species divergence

Evolutionary Rates

-The evolutionary distances between various species can be plotted against the time when the species diverged -There is always a constant mutation rate with respect to time, not generation -The differences in rates at which these mutations are accumulated are the slopes of each line -Defective and advantageous mutations sort themselves based on whether or not they benefit the organism -This is based on the tolerance of change in protein function -Unit evolutionary period is defined as the time required for the amino acid sequence of a protein to change by 1% after two species have diverged -Observe: -Fibrinopeptides evolve quite rapidly as compared to histones, which are highly evolutionarily conserved-relatively little selective pressure on them to maintain their amino acid sequence and thus their rate of variation is high -Histones serve a very fundamental purpose and are well adapted-peas and cows differ by 2 conservative changes in the 102 aa protein-highly intolerant to mutational change

Purification

-The goal: be able to study a single species in a test tube, with other cellular components removed. This allows for you to manipulate the species of interest and change its conditions. -Cellular assays may be more biologically relevant, but oftentimes co-factors can cause confounding issues. -The cell contains thousands of different substances that may have similar properties to the particular molecule you wish to study -A macromolecule of interest might comprise just 0.1% of cellular mass, but purity is desired to be >99% - it is required to get your protein to 98-99% purification to really have solid assay data -This is a fractionation process-knowing a bit about the properties of the protein, these properties can be used to isolate the protein of interest from the cellular millieu -If a protein has cofactors-you need to separate the protein from these so that you you can analyze it on its own -Allows you to manipulate conditions-if you want to know how a protein functions under different conditions- you can't do this in the cell

Exclusion limit

-The molecular mass of the smallest molecule unable to penetrate the pores of a given gel in gel filtration chromatography -To some extent this value is a function of molecular shape-elongated molecules are less likely to penetrate a given gel pore than spherical molecules of the same molecular volume

Globin Family of Proteins

-There are many forms of hemoglobin expressed at different times during development -These different globin genes are paralogs, related by gene duplication events and in the same organism -It is unlikely that globin genes coevolved to happen to have the same sequence and function -More likely explanation is to assume they started from one gene that likely got duplicated at a point in time -The two genes after duplication can diverge independently -⍺-globin and myoglobin are two fairly different proteins but they are still considered paralogs -Exolutionary relationship between these genes is described in the gene tree

Electrophoresis

-This is the migration of ions in an electric field which is widely used for the analytical separation of biological molecules -Used to assess the success of the purification at each step and for experimental purposes -In a Gel, this is the migration of ions in an electric field and through a gel matrix -Proteins: polyacrylamide gels -Nucleotide: agarose gels (allow for separation of much larger molecules -Setup consists of a gel, cathode and anode connected to two different sides -Samples are added to the top wells -Current is provided, separating the samples by weight-comparing the bands in the gel to a ladder that tells you the size

Invariant Residues

-When the same side chain is at a particular position in the amino acid sequence of a series of related proteins -The chemical and/or structural properties of this residue uniquely suit it to some essential function of the protein

Primary Structure

-the amino acid sequence of its polypeptide chain(s) and for a nucleic acid is its base sequence Importance: -The knowledge of a protein's amino acid sequence is essential for an understanding of its molecular mechanism of action -Prerequisite for the elucidation of its three-dimensional structure by both X-ray crystallography and NMR spectroscopy - Sequence comparisons among analogous proteins from the same individual, from members of the same species, and from members of related species have yielded important insights into how proteins function and have indicated the evolutionary relationships among the proteins and the organisms that produce them -Have important clinical applications-many inherited diseases are caused by mutations leading to an amino acid change in a protein

Tertiary Structure

-the three-dimensional structure of an entire polypeptide or polynucleotide chai -The distinction between secondary and these structures is vague; in practice, the term "secondary structure" alludes to easily characterized structural entities such as helices

Example Workflow: Expression

1. Clone with a 6-His tag in frame with the Hsp104 gene 2. Transform the plasmid into bacteria, select a colony that grows on ampicillin supplemented media to ensure the cells have the plasmid with gene of interest 3. Grow the cells, starting from 1 colony to 6L 4. Once the bacteria are growing exponentially, induce expression with IPTG 5. Allow expression to proceed overnight(Avoid overexpressing the protein- once you find a stopping point freeze the cells)

Example Workflow: Purification

1. Harvest the 6L of culture in a large centrifuge 2. Lyse the cells using lysozyme and a sonicator 3. Pellet the cell debris in a centrifuge, leaving the protein in solution 4. Apply the supernatant to Ni-NTA resin to bind the His-tagged protein 5. Following several washes, elute the protein with imidazole 6. Assess Purity via SDS-PAGE-separate slide 7. The protein still has some contaminants, so apply the eluted protein to an anion exchange column-deparate slide, assess purity again with SDS-PAGE *** If you want to purify even further, say for crystallization, perform size exclusion chromatography(separate slide) 8. The protein binds the column because in a low salt buffer 9. Wash off contaminants and elute with a salt gradient 10. Buffer exchange the protein via dialysis to lower salt concentration appropriate for longer term storage 11. Optional: remove the His-tag using TEV protease 12. Quantify the protein, confirm activity 13. Flash freeze the protein using liquid nitrogen-if you freeze in a typical freezer the water molecules will denature the protein

Example Workflow-Cloning

1. Obtain a plasmid from another researcher. This may or may not be in the proper expression vector. Alternatively, obtain yeast genomic DNA(Because this is Baker's yeast, this is trivial) 2. Amplify the Hsp104 sequence via PCR. The primers must incorporate both the N and C terminal regions of Hsp104 - Clone with a 6-His tag in frame with the Hsp104 gene 3. Insert the sequence into an E. coli expression vector and transform/select in E. coli cloning strain 4. Use Sanger sequencing to confirm the cloning is correct 5. Transform the proper plasmid into E. coli strain for expression/purification

Basic Procedure for Primary Structure Determination

1. Prepare the protein for sequencing -Determine the number of polypeptide chains in the protein-need to separate to be able to analyze them individually -Cleave disulfide bonds -Separate and purify the subunits 2. Sequence the polypeptide chains -Fragment-just like in DNA sequencing- the subunits to yield small peptides -Separate and purify the fragments -Determine the amino acid sequence of the fragment -Repeat with a different fragmentation process to cleave at different positions -Done multiple times to get overlapping fragments 3.Organize the complete structure -Span the cleavage points and arrange in order -Determine the positions of disulfide bonds

DNA Microarrays

Addressing such questions as: 1. What are the functions of the genes? 2. What is the expression profile (in which cells, under what circumstances, and to what extent are each of the genes expressed)? 3. What are the medical consequences of variant genes? -The traditional method of the one-gene-at-a-time approach is simply incapable of acquiring the vast amounts of data necessary to answer these questions -What is required are techniques that can simultaneously monitor all the components of a biological system at once-this technique is one -With this, you can compare the expression between samples prepared from different sources -Using these, you can compare the expression of genes in different tissues -Consequently, these hold enormous promise for understanding the interplay of genes during cell growth and changes in the environment, for the characterization and diagnosis of both infectious and noninfectious diseases

Cyanogen Bromide (CNBr)

Cleaves proteins specifically after Methionine residues

Detection of Gel Electrophoresis Bands

Following gel electrophoresis, the proteins in the gel must be stained or otherwise processed to visualize the protein Detection of protein size(do not confirm Identity) -Coomassie blue is most common for abundant proteins -Silver stain is similar to coomassie but 50 times more sensitive --These only tell you how pure the sample is and if the protein of a given size is present-both are nonspecific and just bind to the backbones of the protein Detection of Protein size and Confirmation of Identity -Immunoblot (Western blot) detects trace amounts of specific proteins-requires an antibody to the protein or the tag -Does not provide information regarding purity

Paralogs

Homologous genes within a single species

Interactions That Stabilize Proteins

Hydrophobic effect-most important -The release of water molecules from the structured solvation layer around the molecule as protein folds from a denatured state increases the net entropy -Amino acids in the core drive this process Hydrogen bonds -Interaction of amide and carbonyl- N&C terminus-of the peptide bond leads to local regular structures such as a helices and b sheets. London dispersion(not very imp) -Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein. Electrostatic interactions -Long-range strong interactions between charged groups-opposite charges -Salt bridges strongly stabilize the protein