Chapter 4 and 5

amino acids are

"chiral" --> tetrahedral (sp3) carbons have 4 different substituents (C=chiral center) -associated with 2 enantiomers (enantiomers have otherwise identical physical and chemical properties, unless probed asymmetrically) -all natural L-amino acids=(S-amino acids) (other that cystein bc of its side chain) -figure out stereochemistry of amino acids! Look at professor Deaks' notes using... SH>OH>NH2>COOH>CHO>CH2OH>C6H5>CH3>H (order of priority groups) -atoms of higher atomic number rate higher than atoms of lower atomic number

key concepts:

- environmental conditions such as pH and temp affect a protein's stability during purification -an essay based on a protein's chemical or binding properties may be used to quantify a protein during purification -fractionation procedures take advantage of a protein's unique structure and chemistry in order to separate it from other molecules -increasing salt concentration causes selective "salting out" (precipitation) of proteins with different solubilities -protein's ionic charge, polarity, size, + ligand-binding ability influence its chromatographic behavior -gel electrophoresis and its variations can separate proteins according to charge, size, + isoelectric point -overall size and shape of macromolecules + larger assemblies can be assessed through ultracentrifugation

how are amino acid names abbreviated?

-Glx=glu or gln -Asx=asp or Asn -amino acid residues in polypeptides=drop the suffix, usually -ine, in the name of amino acid + replacing it by -yl. -described by started at N-terminus + proceeding to C-terminus

SDS-PAGE separates proteins by mass

-SDS-PAGE: interfere with hydrophobic interactions that normally stabilize proteins -separates proteins purely by gel filtration effects (according to molecular mass) -relative mobilities of proteins vary linearly with logarithm of their molecular masses -the possibility that subunits are linked by disulfide bonds can be tested by preparing samples for SDS-PAGeE in the presence of absence of a reducing agent that breaks those bonds

how are amino acids dipolar ions? why can they act as acids of bases ? What are dipolar ions?

-amino + carboxylic groups=readily ionize -at physiological pH (7.4), amino groups are protonated and the carboxylic acid groups are in their conjugate base (carboxylate) form --> so amino acids can act as acid or base dipolar ions: molecules (like amino acids) that bear charged groups of opposite polarity -amino acids=ionic, so more soluble in polar substances

how can amino acids be depicted? What stereochemical configuration do all amino acids have?

-amino acids + many other biological compounds are chiral molecules whose configurations can be depicted by Fischer projections -amino acids in proteins all have the L stereochemical configuration

What are other roles of amino acids in metabolism?

-building blocks for proteins but also... -some=certain hormones (important for gland function, diet, etc --> think thyroxine) -neurotransmitters - facilitate chemical communication between 2 neurons (via electrical signal) ... charge imbalance creates "excitement" of 2nd neuron (signal that facilitates transfer=neurotransmitters) --> neurotransmitters=derived from amino acids -amino acids repurposed for many other reasons

Are amino acid chains nonpolar, polar, or charged? what are some details on nonpolar amino acid side chains?

-can be all 3... classified by 1) those w nonpolar R groups 2) those w uncharged polar R groups 3) those w charged polar R groups -nonpolar amino acid side chains have a variety of shapes + sizes -glycine-smallest possible side chain (H) -alanine, valine, leucine, isoleucine=alphatic hydrocarbon side chains ranging from methyl group (alanine) to isomeric butyl groups (leucine + isoleucine) -methionine: thioether side chain -proline: cyclic pyrrolidine side group -phenylanine + tryptophan=contain aromatic side groups

what do proteins evolve from?

-duplication of genes or gene segments -analysis of a large # of proteins indicates that evolutionarily conserved sequences are often segments of about 40-200 residues (domains) -domains whose sequences are more than about 40% identical usually have the same function -domains with less than about 25% sequence identity usually perform different roles

selenocysteine

-e- rich, so very nucleophilic... targets stop codons + repurposes UGA stop codon for incorporating issues; -when ribosome comes to UGA/TGA -all other proteins in cell that end w TGa... how to make them stop (still use stop codon) + not add selenocysteine? -SECIS sequence- present for only mRNA that require incorporation of selenocysteins residue in 3' UTR of mRNA --> binding protein recruites Sec binding factor --> binds tRNA of selenocysteine (only recruited for mRNAs with SECIs incorporated sequence) -elongation factor binds to all tRNAs... but selenocysteine has own specific elongation factor (recruited specifically to mRNAs that need the amino acid seleeocysteine)

what do protein sequences reveal?

-evolutionary relationships --> primary structures of given protein from related species closely resemble one another -related species have evolved from common ancestor, so follows that the genes specifying each of their proteins must likewise have evolved from the corresponding gene in that ancestor

invariant residue? conservatively substituted positions? hypervariable positions?

-finding same residue at particular position in amino acid sequence of series of related proteins suggests that chemical/structural properties of invariant residue=uniquely suit it to some essential function of the protein -conservatively substituted positions: when amino acid positions have less stringent side chain requirements + accommodate residues with similar characteristics -hypervariable position: if particular amino acid position can tolerate many different amino acid residues, indicating that the functional requirements of that position are non-specific

What can protein families arise through?

-gene duplication -homologous proteins with the same function in different species=orthologous -within a species, similar proteins arise through gene duplication (genetic recombination event in which 1 member of a chromosome pair acquires both copies of primordial gene) -gene duplication is a particularly efficient mode of evolution bc 1 copy of the gene evolves a new function through natural selection while its counterpart continues to direct the synthesis of the original protein -paralogous: 2 independently evolving genes that are derived from duplication event

two-dimensional gel electrophoresis

-sample of proteins subjected to IEF in one direction, + separated proteins subjected to SDS-PAGE -generates an array of spots, each representing a protein -valuable tool for proteomics --> individual protein spots can be excised + protein identified by mass spectrometry (detects variations in positions + intensities of protein spots in samples obtained from different tissues or under different growth conditions)

What do chiral centers give rise to?

-give rise to enantiomers -enantiomers=molecules that are nonsuperimposable mirror images --> only when probed asymmetrically, for example, by plane-polarized light or by reactants that also contain chiral centers, can they be distinguished or differentially manipulated -not possible to predict optical rotation from the structure of a molecule or to derive the absolute configuration (spatial arrangement) of chemical groups around a chiral center from optical rotation measurements

What are some details on uncharged polar side chains?

-have hydroxyl, amide, or thiol groups -serine + threonine=hydroxylic R groups of different sizes -asparagine + glutamine=amide-bearing side chains of diff sizes -tyrosine=phenolic group -cysteine=thiol group that can form disulfide bond w another cysteine through oxidation of 2 thiol groups

what is life made of? what can happen after amino acids have been made?

-life is made of chiral molecules, and chirality has important biological consequences (ex: ibuprofen --> only specific enantiomer has anti-inflammatory action) -post-translational modifications of amino acids can occur --> when initially synthesized, have limited 20 amino acids ... but once protein made (after translation) can be chemically modified (installing new chemistries onto proteins)

What can limited proteolysis yield?

-limited proteolysis (by adjusting reaction conditions + limiting reaction times, less specific endopeptidases can yield a set of discrete, nonoverlapping fragments -CNBr: cleaves on C side of Met residues (promote peptide bond cleavage at specific residues)

what are polypeptides? what are large contributors to the diversity in shapes + biological functions of proteins?

-linear polymers (residues at 2 ends=each participate in 1 peptide bond) -residue w free amino group=amino terminus (N-terminus) -residue w free carboxylate group=carboxyl terminus (C-terminus) -variations in the length + amino acid sequence of polypeptides are major contributors to the diversity in the shapes + biological functions of proteins

some amino acids are biologically active

-many amino acids=synthesized to function independently -can be oxidized as metabolic fuels to provide energy -often function as chemical messengers between cells --> ex: GABA + dopamine=neurotransmitters (substances released by nerve cells to alter behavior of neighbors) -histamine=potent mediator of allergic reactions -thyroxine=iodine-containing thyroid hormone that generally stimulates vertebrate metabolism

the Pk of ionizable groups depend on: what is the isoelectric point:

-nearby groups -a-amino acids=2-3 acid-base groups (at very low pH, fully protonated, at very high pH, unprotonated, at intermediate pH, acidic groups unprotonated + basic groups protonated) -in aqueous solutions, the un-ionized form (H2NCH2COOH)=present only in vanishing small quantities pI=1/2(pKi+pKj) --> pH at which a molecule carries no net electric charge Ki+Kj=dissociation constants of 2 ionizations involving neutral species

what links amino acids? what are amino acid residues?

-peptide bonds -polymerized to form chains through condensation --> resulting CO-NH linkage=peptide bond -polymers of 2,3,few amino acid units=dipeptides, tripeptides, oligopeptides, polypeptides (many amino aid units) -when incorporated into peptide, individual amino acids=amino acid residues

What does glutathione do?

-plays role in cellular metabolism --> Glu-cys-gly peptide where Y-carboxylate group of glutamate chain forms an isopeptide bond w amino group of cys residue -2 tripeptides undergo oxidation of SH groups to form dimeric disulfide-linked structure called GSSG (which helps inactivate oxidative compounds that could potentially damage cellular structures)

What happens in mass spectrometry for protein or peptide sequencing? What is it based off of? What is mass spec an important technique for? What does it accurately measure?

-production of ions, that are subsequently separated or filtered according to their mass-to-charge (m/z) ratio and detected. resulting mass spectrum=plot of relative) abundance of the produced ions as a function of the m/z ratio - characterizing and sequencing polypeptides. 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).

What do sequence comparisons provide? what does the comparison of primary structures of homologous proteins provide?

-provide information on protein structure and function -comparisons of primary structures of homologous proteins (evolutionarily related to proteins) indicate which of the protein's residues are essential to its function, which are less significant, and which have little specific function

What does hydrophobic interaction chromatography do?

-purifies nonpolar molecules -matrix material lightly substituted with octyl or phenyl groups --> at high salt concentrations, nonpolar groups on surface of proteins "interact" with hydrophobic groups

What does capillary electrophoresis do?

-readily separates charged molecules -electrophoresis carried out in thin capillary tubes --> readily dissipate heat + permit use of high electric fields (quicker separation)

what does it mean when all of the amino acids recovered from polypeptides are optically active (w exception of glycine?)

-rotate plane of polarized light -polarimeter: measures direction and angle of rotation -optically active molecules=asymmetric (not superimposable on their mirror image) -central atoms in molecules w asymmetric centers=chiral centers (have property of chirality)

salting out proteins by solubility?

-salting in: solubility of protein at low ion concentrations increases as salt is added --> additional ions shield protein's ionic charges + weaken attractive forces between individual protein molecules --> as more salt added, solubility of protein decreases (salting out result of competition between added salt ions + other dissolved solutes for molecules of solvent) -adjusting salt concentration in solution containing mixture of proteins to just below the precipitation point of the protein to be purified eliminates many unwanted proteins from solution --> after removing precipitated proteins by filtration, salt conc of remaining solution is increased to precipitate the desired protein

What does electrophoresis do?

-separates molecules according to charge and size: -PAGE: molecular separations based on get filtration (size + shape) + elecrophoretic mobility (electric charge) -electrophoretic mobility of smaller molecules is greater than mobility of larger molecules with the same charge density -immunoblotting: if protein of interest available, can be used to detect protein on gel in presence of many other proteins

what does gel filtration chromatography do ?

-separates molecules according to size -molecules separated according to their size and shape -if aq solution of molecules of various sizes passed through column containing "molecular sieves," molecules that are too large to pass through pores are excluded from solvent volume inside gel beads --> these large molecules therefore traverse the column more rapidly than small molecules that pass through the pores

protein evolution (4) key concepts

-sequence comparisons reveal the evolutionary relationships between proteins -protein families evolve by the duplication and divergence of genes encoding protein domains -the rate of evolution varies from protein to protein evolution: when mutation arises that improves fitness of host under prevailing conditions

How do the 20 amino acids vary? what kinds amino acid residues? what do some amino acid side chains contain?

-the 20 amino acids (that all proteins composed of) share a common structure but differ in their side chains -peptide bonds link amino acid residues in a polypeptide -some amino acid side chains contain ionizable groups whose pK values may vary

Sanger sequencing... what must the protein be broken down into?

-the protein must be broken down into fragments small enough to be individually sequenced, and the primary structure of the intact protein is then reconstructed from the sequences of overlapping fragments

Info about amino acid derivatives?

-the side chains of amino acid residues in proteins may be covalently modified -some amino acids and amino acid derivatives function as hormones + regulatory molecules

Protein sequencing key concepts (3)

-to be sequenced, a protein must be separated into individual polypeptides that can be cleaved into sets of overlapping fragments -the amino acid sequence can be determined by Edman degradation, a procedure for removing N-terminal residues one at a time -mass spectrometry can identify amino acid sequences from the mass-to-charge ratio of gas-phase protein fragments -protein sequence data are deposited in online databases

what does the size range reflect?

1) 40 residues appear to be near the minimum for a polypeptide chain to fold into a discrete and stable shape that allows it to carry out a particular function 2) polypeptides with well over 1000 residues may approach the limits of efficiency of the protein synthetic machinery --> the longer the polypeptide (+ longer mRNA), greater likelihood of introducing errors during transcription and translation -each amino acid residue has characteristic chemical + physical properties -hydrophobic residues in interior + hydrophilic side chains on surface

Book continued: Amino acid sequences are valuable because:

1) knowledge of a protein's amino acid sequence is prerequisite for determining its 3D structure + is essential for understanding its molecular mechanism of action 2) sequence comparisons among analogous proteins from different species yield insights into protein function and reveal evolutionary relationships among the proteins and the organisms that produce them 3) many inherited diseases are caused by mutations that result in an amino acid change in a protein --> amino acid sequence analysis can assist in the development of diagnostic tests and effective therapies

What is the first step of protein sequencing?

1) separate subunits --> N-terminal analysis reveals the number of different types of subunits - identifying the "end group" can establish the number of chemically distinct polypeptides in a protein -fluorescent compound 5-dimethylamino-1-napthalenesulfonyl chloride polypeptides -treatment of dansylated polypeptide w aq acid at high temperature hydrolyzes its peptide bonds --> liberates dansylated N-terminal residue, which can then be separated chromatographically from the other amino acids (identified by fluorescence)

for a protein of n residues there are... how many residues do most polypeptides contain?

20^n possibly sequences -polypeptides smaller than 40 residues=peptides -largest=titin -majority of polypeptides contain 100 and 1000 residues -multisubunit proteins= contain several identical and/or nonidentical chains called subunits

pK for a-amino groups in proteins?

7.5-8.5 -in free amino acids, pK values are higher, due to the electron-withdrawing character of nearby carboxylate group (which makes it more difficult for ammonium group to become deprotonated)

What happens during edman degradation? How many residues can it sequence? what are the challenges?

After phenyl isocyanate (PITC) mediated cleavage of the N-terminal residue, next residue at the N-terminus is exposed, which can be subsequently sequenced Modern automated Edman sequencing can routinely sequence ~50 residues with 99% accuracy; up to ~100 residues can be sequenced Challenges: If the N-terminus is capped, it won't work. Reaction stops at non-α-amino acid centers such as iso-aspartic acid

How do you efficiently purify a target protein?

Affinity chromatography -fusion protein (on C or N terminus) binds with GST example: GST GST-tagged proteins bind to gluthatione on beads Non-specifically or weakly bound proteins washed off GST-tagged proteins eluted with glutathione (competitor) or thrombin (protease) -bring all proteins to center w GST... then unbind + sent it out -add protease splitting site between GST and target protein (adds more attraction to remove GST)

within ion exchange chromatography, how do the beads work? what do different proteins bind with? what is it eluted with? Different proteins elute at different...

Beads have charged group: + charge binds acidic amino acids - charge binds basic amino acid Different proteins bind with different affinity Eluted with increasing amount of salt (NaCl or KCl) Different proteins elute at different salt concentrations DEAE: matrix-CH2-CH2-NH(CH2CH3)2+ --> interact more strongly with negatively charged proteins CM: matrix-CH2-COO- --> interact more strongly with positively charged proteins

protein purification by chromatography:

Dramatically reduces the complexity associated with protein purification Any expressed protein can be purified; regardless of its activity

D-amino acid residues

D-amino acid residues=components of some relatively short bacterial polypeptides --> presence of D-amino acids renders bacterial cell walls less susceptible to attack by peptidases that are produced by other organisms to digest bacteria

What is a major measurement of enzyme activity? What are easy to analyze? What is a coupled enzymatic reaction? what are immunoassays based on? RIA vs ELISA?

Enzymes that catalyze reactions with readily detected products are easy to analyze. Substances with colored or fluorescent products have been developed for just this purpose coupled enzymatic rxn: If no such substance is available for the enzyme being assayed, the product of the enzymatic reaction may be converted, by the action of another enzyme, to an easily quantified substance. Immunoassays based on antibodies that specifically bind the target protein. Radioimmunoassay (RIA), or enzyme-linked immunoassay (ELISA)

Salting out: what does a protein solubility depend on? Can these be manipulated?

First line of purification to reduce complexity of sample: Because a protein contains multiple charged groups, its solubility depends on the concentrations of dissolved salts, the polarity of the solvent, the pH, and the temperature. Some or all of these variables can be manipulated to selectively precipitate certain proteins while others remain soluble. -salt of choice (ammonium sulfate) added to soln of proteins to conc just below the precipitation point of the protein of interest -after centrifugation, unwanted precipitated proteins (Red) are discarded + more salt is added to supernatant to concentration sufficient to salt out the desired protein (green) -after second centrifugation, protein recovered as a precipitate + supernatant is discarded. -add salt --> interacts strongly with polar water --> organic molecule then "salts out" of solution -have precipitate + supernatant --> find what protein was in the precipitate + what still in supernatant --> if still in supernatant, progressively increase the concentration of salt (so that eventually precipitates out)

What is Ni-NTA chromatography?

Hexa-histadine tag is small, yet allow efficient purification of almost any protein -Ni/Co- 6 coordination sides (4/6=bound to matrix) --> immobilized metal ion column used to purify proteins (just with 6 histadines in a row) -how to elute protein (kick out of column)? can use imidazole, change pH to acidic (pH=5.... N will deprotonate + be unable to bind to Ni), or use a strong chelator that removes Ni + 2.

What is heterologous protein expression?

Instead of dealing with a biological system that is difficult to manipulate, use an easy "expression host" to make your protein • High degree of control over the timing and level of expression of the target protein • High degree of control over the sequence of the protein expressed hierarchy of expression hosts: bacteria, yeast, easy to grow insect cells (SF9), easy to grow mammalian cells (mouse, human,) native tissue recombinant plasmid technology --> complementation into bacteria... if too big, organism may need help (higher order organisms proteins may not be folded right in a lower organism)

What is necessary after a pure protein is obtained in order to establish its identity? What is this done through? Why sequence proteins, when you can simply infer their sequences from the available genome sequences?

Once a pure protein is obtained, it is necessary to determine its primary sequence to establish its identity --> protein sequencing - Not all open reading frames (ORFs) are accurately predicted by the algorithms used to interpret genome sequences - From the same ORF, multiple different proteins can be produced by alternative splicing - Proteins can undergo post-translational modifications, which can only be identified through direct chemical characterization

how are proteins purified through chromatography? what can molecules be separated on the basis of?

Protein mixture applied to column - Solvent (buffer) applied to top, flowed through column - Different proteins interact with matrix to different extents, migrate at different rates - Proteins collected separately in different fractions -can separate 2 different compounds separated on basis of: -charge (ion exchange) --> different proteins have different overall charge -hydrophobicity/polarity- hydrophobic interaction chromatography -size- gel filtration --> bead has pores

How can proteins be damaged? How can proteins aggregate? What do some require? For long-term storage, what must proteins be stored at?

Proteins can be damaged through oxidation by molecular oxygen; limit exposure and use reducing agents such as beta-mercaptoethanol Proteins can aggregate through hydrophobic patches on surface. May require the presence of detergents to stabilize them For long-term storage, protein must be stored at very low temperatures (-80 to -196 °C), in the presence of cryo-protectants and reducing agents

What are challenges with classical protein purification?

Requires an unique activity of the target of protein to identify it in the fractions: Proteins with unknown activities are difficult to purify • Extremely time consuming • Not suitable for unstable proteins • Not scalable; limited by its abundance in natural sources • Weakly or conditionally expressed proteins are difficult to obtain by this method • Only the natural isoforms of a protein can be purified

What are chiral centers? What are they associated with?

Tetrahedral (sp3) carbons that have four different substituents are chiral centers They are associated with two enantiomers. Enantiomers have otherwise identical physical and chemical properties, unless probed asymmetrically Order of priority of functional groups: SH>OH>NH2>COOH>CHO>CH2OH>C6H5>CH3>H

Characterizing purified proteins: Concentration What can the concentration of a substance in soln be measured by? What does a solution containing a solute that absorbs light do so according to? what can the protein's extinction coefficient by calculated from? What is Bradford assay, and what does it use?

The concentration of a substance in solution can be measured by absorbance spectroscopy. A solution containing a solute that absorbs light does so according to the Beer-Lambert law A=log(Io/I)=ECl proteins extinction coefficient can be calculated from the # of these residues per molecule Concentration of any protein can be also measured by Bradford assay, which uses Coomassie blue, a reagent that turns blue when adsorbed onto proteins (not as accurate as absorption spectroscopy, but quicker and easier)

CLASS how should the pH and temperature be maintained? What happens when the target protein is released from cells by "cell-lysis"? What leads to the loss of proteins? what concentration should you keep the protein mixture at ?

The pH and the temperature should be maintained at levels where the protein is stable. Typically pH near 7, temperature near 0 °C When releasing the target protein from the cells by "cell-lysis", it also releases degrading enzymes such as proteases. These must be inactivated to prevent loss of target protein Proteins can be "sticky" and attach to solid surfaces by adsorption, leading to their loss. Keep at high concentration/in the presence of other proteins

3) what happens to the polypeptide chains? what are endopeptidases used for? trypsin? what do endopeptidases exhibit and yield?

The polypeptide chains are cleaved -endopeptidases (enzymes that catalyze hydrolysis of internal peptide bonds, as opposed to exopeptidases which catalyze hydrolysis of N- or C- terminal residues) can be used to fragment polypeptides -both endopeptidases + exopeptidases (proteases)=have side chain residues flanking peptide bond -trypsin: greatest specificity, digestive enzyme -endopeptidases=exhibit broader side chain specificities + yield series of peptide fragments with overlapping sequences

protein purification by chromatography: to purify a protein from a complex mixture, what is attemped? what evaluates the presence of protein in different fractions? After optimization, what happens to isolate a protein with acceptable homogeneity?

To purify a protein from a complex mixture, different fractionation strategies are attempted by trial and error, and the presence of the protein in different fractions are evaluated by activity assays After much optimization, a purification protocol, composed of a series of specific fractionation step is established that isolates the protein with acceptable homogeneity -if proteins going quickly, early fractions -if proteins interact more strongly, later fractions

What happens in analytical centrifugation? what is a protein's sedimentation coefficient expressed using? what do protein complexes range from? viruses? bacterial ribosomes?

When subjected to ultracentrifugation (up to 80,000 rpm) high centrifugal force forces macromolecules to separate by size/molecular weight when subjected to a density gradient (solution of sucrose/CsCl, where the density gradually increases) A protein's sedimentation coefficient (its sedimentation velocity per unit of centrifugal force) is usually expressed using the Svedberg (S) units. Protein complexes range from 1-50S; viruses 40- 1000S; bacterial ribosome is 70S, its two subunits are 50S and 30S each Ultracentrifugation can be used for both analytical purposes, as well as for fractionating macromolecules for preparatory purposes

What are the common types of amino acids?

a-amino acids bc have amino group (-NH2) as a substituent of the a carbon atom (C next to -COOH group) -20 amino acids=differ in the structures of their side chains (R groups)

proteins: what do they do? CHAPTER 5 NOTES

all molecular transformations that define cellular metabolism mediated by protein catalysis --> regulatory roles (monitoring extracellular + intracellular conditions + relaying info to other cellular components), essential structural components of cells, etc primary structure: amino acid sequence of polypeptide chain (synthesized in vivo by stepwise polymerization of amino acids in order specified by nucleotides)

What are assays?

assay: must be devised... specific for target protein, highly sensitive, + convenient to use -rate of produce formation is proportional to the amount of enzyme present -coupled enzymatic reaction: when no substance is available for enzyme being assayed ,so produce of enzymatic reaction is converted by action of another enzyme to an easily quantified substance

salting out: fractionating protein based on solubility

bc protein contains multiple charged groups, its solubility depends on the concentrations of dissolved salts, polarity of solvent, pH and temp. Some or all of these variables can be manipulated to selectively precipitate certain proteins while others remain soluble

What are many drugs chemically synthesized as?

chemically synthesized as racemic mixtures, although only 1 entantiomer has biological activity -occasionally, inactive enantiomer of useful drug produces harmful effects + must be eliminated from the racemic mixture (ex: thalidomide)

chromatography involves interaction with mobile and stationary phases:

chromatography: as solutes flow through column, interact with stationary phase + retarded --> substances with different rates of migration separated -HPLC: automated systems with applied samples, controlled flow rates at high pressures, + chromatographic matrix of specifically fabricated glass/plastic beads coated w chromatographic material -> improves speed/resolution/reproducibility of separation

What does the fischer convention describe?

configuration of asymmetric centers -fischer projections: horizontal bonds=extend above plane of paper + vertical bonds=extend below plane of paper -all amino acids derived from proteins have the L stereochemical configuration; same relative configuration around their Ca atoms -molecule w n chiral centers=2^n different possible stereoisomers

Step 2)

disulfide bonds between and within polypeptides are cleaved -disulfide bonds between Cys residues must be cleaved to separate polypeptide chains (if disulfide-linked) + to ensure that polypeptide chains are fully linear -disulfide bonds can be reductively cleaved by treating them w a mercaptan (-SH) -resulting free sulfhydryl groups=alkylated (iodoacetate) to prevent the reformation of disulfide bonds through oxidation w O2

What does affinity chromatography exploit?

exploits specific binding behavior --> characteristic of proteins=ability to bind specific molecules tightly but noncovalently -ligand that specifically binds to protein of interest is covalently attached to an inert matrix -when an impure protein solution is passed through this chromatographic material, desired protein binds to the immobilized ligand, whereas other substances are washed through the column with the buffer -desired protein can be recovered in highly purified form by changing elution conditions to release the protein from the matrix -immunoaffinity chromatography: antibody attached to matrix in order to purify the protein against which the antibody was raised -metal chelate affinity chromatography: divalent metal ion (Zn2+ or Ni2+) is attached to the chromatographic matrix so that proteins bearing metal-chelating groups can be retained -recombitant DNA techniques used to append a segment of 6 consecutive residues (His tag) to N- or C- terminus of the polypeptide to be isolated

what are proteins purified by?

fractionation procedures --> eliminate selectivity of other components of the mixture so that only the required substance remains

What is capillary electrophoresis? What disadvantages does it overcome?

gel electrophoresis=highly effective at separating charged molecules, but slow + difficult to quantitate and automate. capillary electrophoresis (CE)-electrophoresis is carried out in very thin capillary tubes. narrow capillaries rapidly dissipate heat and hence permit the use of very high electric fields, which reduces separation times to a few minutes.

globin family? hemoglobin? myoglobin?

globin family of proteins: provides excellent example of evolution through gene duplication divergence -hemoglobin: transports O2 from lungs (tetramer, a2B2) -sequences of a and B subunits are similar to each other + to sequence of myoglobin (facilitates oxygen diffusion through muscle tissue) -pseudogenes (globin genes that aren't expressed)=dead ends of protein evolution --> duplicated gene only has limited time to evolve new functionality that provides selective advantage to its host before inactivated through mutation + becomes pseudogene

fluorescent protein PTMS:

if combine peptides together, can? get fluorescence (maybe binding specific molecule?) -post-translational modification: PTM react with each other + form structure that creates di-aromatic functionality (2 cyclic structures connected to double bond) --> all electronically connected, + leads to fluorescence -green fluorescent protein-- 3 PTM residues all interact (1st=condensation, 2nd=oxidation)

protein side chains may be modified...

in almost all cases, unusual amino acids result from the specific modification of an amino acid residue after the polypeptide chain has been synthesized

ion exchange chromatography separates anions + cations

ion exchange chromatography: charged molecules bind to oppositely charged groups that are chemically linked to matrix -anions bind to cationic groups on anion exchangers -cations bind to anionic groups on cation exchangers -DEAE: most frequently used anion exchanger -CM: most commonly used cation exchanger

mass spec formats:

ionization source: MALDI, Electrospray mass analyzer: TOF, Linear quadrupole ion trap, FT-ICR detector: • Electron multiplier

what determines the molecular masses of peptides?

mass spectrometry- measures mass to charge ratio for ions in gas phase ESI: macromolecule sprayed from narrow capillary tube maintained at high voltage --> charges result from protonation of basic side chains like Arg + Lys -short polypeptides (<25 residues)=directly sequenced through use of tandem mass spectrometer -by comparing molecular masses of successively larger members of a family of fragments, the molecular masses + therefore the identities of the corresponding amino acid residues can be determined -reconstructed protein sequences are stored in databases

selenocysteine is ...

more nucleophilic and redox-active than cysteine cysteine: TGT + TGC codons selenocysteine: TGA codon -much more acidic, so more efficient nucleophilic attack (highly negatively charged, so susceptible to be oxidized) -selenium=more readily oxidized and more nucleophilic --> if have disulfide trying to break, Se is a more efficient nucleophile (more readily oxidized) ... more efficiently reduces disulfide bonds -selenium in binding side --> more efficient nucs + catalyzing reactions other examples other than selenoproteins: glutathione peroxidase + thioredoxin reductase

What must be controlled in order to keep proteins stable?

pH and temp; pH: biological materials dissolved in buffer solutions effective in pH range in which materials are stable (failure to do so could cause denaturation/structural disruption) temperature: most proteins denature at high temperatures (so protein purification normally carried out at temps near OC) presence of degradative enzymes: proteases + nucleases --> can be inhibited by adjusting pH/temp to values that inactivate them or adding compounds to block their action absorption to surfaces: many proteins denatured by contact with air-water or glass/plastic surfaces Long-term storage: other processes like slow oxidation + microbial contamination must be prevented

Class; pkas are highly....

pKas are highly context dependent - Electrostatic effects - H-bonding effects - Inside a protein: more difficult to ionize a residue (e effects)

What are some details on charged polar side chains?

positively or negatively charged (side chains of basic amino acids=positively charged at physiological pH values) lysine: butylammonium side chain arginine: guanidino gorup histidine: imidazoium moiety aspartic acid + glutamic acid (side chains of acidic amino acids)=negatively charged above pH=3) -in ionized state, often referred to as aspartate + glutamate) -structures of most polypeptides depend on tendency for polar + ionic side chains to be hydrated + for nonpolar side chains to associate w each other rather than w water (hydrophobic effect)

proteins primary structure... what is the sequence space? size of proteins? are all amino acids respresented equally?

primary structure: amino acid sequence of polypeptide chain -"sequence space" for peptides=astronomically large -in natural proteins, only tiny fraction of sequence space is represented -size of proteins are highly variable (most=100-1000 amino acid residues long) -not all amino acids are represented equally. most abundant: Leu, Ala, gly, val, glu, ser (rarest=trp, cys, met, his)

rate of protein evolution also depends on

protein's structural stability -DNA sequences control expression of proteins, + can also be mutated (control where, when + how much of the corresponding protein is made) -many proteins contain domains that occur in other proteins --> sequence identity between homologous domains is imperfect bc each domain evolves independently

what is a racemic mixture?

racemic mixture: containing equal amounts of each enantiomer -biosynthetic processes=almost invariably produce pure stereoisomers -since most biological molecules are chiral, given molecule will bind to or react w a single enantiomer of another compound

What is neutral drift? what are phylogenetic trees?

random nature of mutational processes change a protein in ways that don't significantly affect its function (neutral drift, over time) phylogenetic tree: diagram that indicates the ancestral relationships among organisms that produce a protein

What is edman degradation?

removes peptide's first amino acid residue: -PITC reacts with N-terminal amino group of polypeptide under mildly alkaline conditions to form PTC adbuct --> -product treated with trifluoroacetic acid (cleaves N-terminal residue) -thiazolone-amino acid --> converted to PTH with aq acid 3 stages... amino acids residues can be sequentially removed from N-terminus of polypeptide in controlled way -releases N-terminal amino acid residue but leaves intact rest of polypeptide chain -possible to determine the amino acid sequence of a polypeptide chain from the N-terminus inward by subjecting the polypeptide to repeated cycles of Edman degradation and, after every cycle, identifying the newly liberated PTH-amino acid

what does two-dimensional electrophoresis do?

resolves complex mixtures of proteins -if a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode, each protein will migrate to the position in the pH gradient corresponding to its pI -IEF: each species of protein "focused" into narrow band about its pI

What does Two-dimensional gel electrophoresis resolve? what is isoelectric focusing?

resolves complex protein mixture -proteins separated by native charge (no SDS) by isoelectric focusing, then denaturing (+SDS) PAGE to separate them by size Isoelectric focusing (IEF): If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode, each protein will migrate to the position in the pH gradient corresponding to its pI.

What does SDS-page do?

resolves proteins based solely on its size -sample loaded into gel -protein w 2 subunits joined by disulfide bridge -heated with SDS + mercaptoethanol -gel electrophoresis

What does utracentrifugation separate?

separates macromolecules by mass -only when subjected to enormous accelerations do macromolecules begin to sediment -ultracentrifuge: demonstrated that proteins are macromolecules with homogeneous compositions + that many proteins contain subunits -the rate at which a particle sediments in the centrifuge is related to its mass (S) today: sedimentation carried out in solution of an inert substance in which the concentration (+ density) of solution increases from top to bottom of centrifuge tube (Density gradient) --> during centrifugation, each species of macromolecule moves through performed gradient at rate largely determined by its sedimentation coefficient + travels as a zone that can be separated from other zones

What is ion exchange chromatography? What kinds of charges do proteins bear? for each protein there is a unique ___. what is this called? What is the net charge here? when does protein solubility tend to decrease?

separation by charge Proteins and other polyelectrolytes (polyionic polymers) bear both positive and negative charges. Depending on the distribution of amino acid residues, for each protein there is a unique pH, where its net charge is 0. This is called the isoelectric point, and the corresponding pH is called pI (measure using isoelectric point, PI) --> higher PI=positive charge, lower PI=lots of aspa + glutate, - charge Protein solubility tends to decrease right around their isoelectric point -if slowly lowered pH, overall negative charge would balance out positive charge elsewhere --> Fnet=0 (pI<7 so that charge becomes 0) -pI=indicator of whether or not protein likes to be + or - charged

size exclusion chromatography?

separation by size, gel-filtration chromatography Beads have different size pores As column flows: • large proteins excluded from pores and therefore flow rapidly • small proteins enter pores and flow slowly

how do proteins differ from each other?

size, hydrophobicity/hydrophilicity, charge, solubility (when a protein is folded, how it interacts w outside environment, etc)

What can protein concentrations be determined by?

spectroscopy- measures concentration of substance in solution Beer lambert law: A=log(Io/I)=Ecl A=absorbance I0=intensity of incident light at any given wavelength E=absorptivity of solute at wavelength C=concentration l=length of light path in cm absorption spectrum=plot of A or E versus wavelength -if a protein has a chromophore that absorbs in visible region of spectrum, this absorbance can be used to assay for the presence of protein in mixture of other proteins

What does the binding affinity depend on?

the binding affinity of a particular protein depends on the presence of other ions that compete with the protein for binding to the ion exchanger and on the pH of the solution, which influences the net charge of the protein -proteins to be separated/dissolved in buffer of app salt+pH concentration applied to column with ion exchanger --> washed with bugger (proteins w low affinities for ion exchanger move through column faster) --> proteins that bind tightly to ion exchanger=eluted by applying buffer (eluant w higher salt concentration of pH that reduces affinity with which the matrix binds to the protein)

How can IEF be combined with SDS-page? how many proteins can be resolved by a single gel? what can individual protein spots in a stained 2D gel be excised with ? what can the protein be characterized by?

two-dimensional (2D) gel electrophoresis. -sample of proteins is subjected to IEF in one direction, + separated proteins subjected to SDS-PAGE in the perpendicular direction. Up to 5000 proteins can be resolved valuable tool for proteomics. Individual protein spots in a stained 2D gel can be excised with a scalpel, destained, and the protein can be characterized by mass spectrometry

protein purification: classic protein purification what are some considerations for this? look at picture on powerpoint!

understanding the properties of a protein requires the ability to purify them to near homogeneity -classical protein purification: using combination of successive rounds of different chromatography, target protein exhibiting an activity can be isolated --> traditionally, this is how proteins isolated for biochemical analysis considerations from complex mixture: -pH and temp should be maintained at levels where protein is stable (ph near 7 and temp near 0C) -when releasing target protein from cells by "cell-lysis," also releases degrading enzymes like proteases... must be inactivated to prevent loss of target protein -proteins can be "sticky" (hydrophobic patches) + attach to solid surfaces by adsorption, leading to their loss (keep at high conc/in presence of other proteins) -proteins can be damaged through oxidation by Oxygen (residues sensitive to O2); limit exposure + use reducing agents like beta-mercaptoethanol -proteins can aggregate through hydrophobic patches on surface... may require presence of detergents to stabilize them --> store at low temps

What are immunoassays?

use antibodies (proteins produced by animal's immune system in response to introduction of foreign substance/antigen) RIA: protein indirectly detected by determining degree to which it competes with radioactively labeled standard for binding to antibody ELISA: many variations

The rate of sequence divergence....

varies -rate at which mutations are accepted into a protein varies -Histone H4: protein that binds to DNA in eukaryotes --> highly conserved protein, extremely intolerant of any mutations -fibrinopeptides- 20 residue fragments long, cleaved from vertebrate protein fibrinogen to induce blood clotting (once removed, fibrinopeptides=discarded so subject to little selective pressure to maintain amino acid sequnces)

To be sequenced, what must a protein be separated into? What can the amino acid sequence be determined by? what can mass spectrometry identify, and how?

• To be sequenced, a protein must be separated into individual polypeptides that can be cleaved into sets of overlapping fragments. • The amino acid sequence can be determined by Edman degradation, a procedure for removing N-terminal residues one at a time. • Mass spectrometry can identify amino acid sequences from the mass-to-charge ratio of gas-phase protein fragments. • Protein sequence data are deposited in online databases and further validated by comparing to those predicted by genome sequences