Biochemistry Lab Final

what is Km?

(k-1+k2)/k1; for rapid equilibrium enzymes (enzymes where k-1 >> k2): Km=(k-1+k2)/k1 we can assume k2 is negligible so it becomes k-1/k1; rate constant for formation of ES=k1[E][S], rate of dissolution of ES is =k-1[ES]; when at equilibrium, two rates are equal -> set them equal to get [E][S]/[ES]=k-1/k1=Keq=Kd (dissociation of ES); so, Km is approx to Kd so it is a measurement of the affinity of the substrate to the enzyme

how do we simplify real complex enzymatic rxn and make it fit MM model? E+S ->(k1) <-(k-1) with ES -> P+E

-assume ES complex forms product and enzyme with only a single rate constant (K2); approximation bc there are many covalent intermediates before getting product, we are saying our kinetics are modeling the ready limiting step (slowest step) - ES going to problem and enzyme is not a reversible reaction, so we will measure initial velocities at the beginning of the reaction (1-9 mins) when less than 10% of substrate has been consumes, so amt of product is low enough to where the reaction is not reversible -amount of ES is approx constant during rxn, steady state assumption -> can make this assumption bc substrate is much more abundant than enzyme so most enzyme-subs is constant, or we can come to this conclusion bc of rapid equilibrium assumption where k-1>>k2 (rxn forming ES is at equilibrium and is much faster than formation of product) -one substrate, not two (true for many enzymes like isomerases; but most are bisubstrate - > include one substrate at very high excess 10x above Km so kinetis is not affected by that substrate bc it is at such large excess)

how did we prepare EcSufS wildtype reaction sample

25uL 10X MOPS, 25uL 500uM cysteine, 1uL of TCEP, fill to 250 uL with water; then add 25uL SufS to get 2.5uM sufE, 0.5 uL to get 0.5 uM SufS wt; exact same method for SufS mutant except to prepare the correct concentration of SufS mutant: 1.16uL in 2000uL of water to get 0.5uM concentration -work for that 860.4uM(x)=0.5uM(2000uL); 860.4 found from beer lambert's law/quantitation experiment

protein quantitation expriments

A280 or bradford assay; use standards to determine concentration or use beer lamberts law

pauling predicted

B sheet - driven by constrictions of planar peptide bonds, only specific phi and psi angles being possible (limit clashes of atoms in background); can be parallel or antiparallel

main findings in Kim et al paper

Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems: studied the regulatory mechanism of SufS shifting between two active sites using backbone amide hydrogen-deuterium exchange mass spec, found that there were 4 peptides exhibiting changes in deuterium uptake upon formation of SufS covalent persulfide intermediate. Previous results point toward an additional level of regulation through a "half-sites" mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities (two active site, and two at dimer interface); residues must form a conduit between the two active sites upon persulfide formation and allows for monomer interaction (half-site regulation); also found three evolutionarily conserved residues at the dimer interface (R92 (on one monomer), E96, and E250(other monomer)) and investigated by alanine scanning mutagenesis, found a 6-fold increases in the value of KSufE for 96 and 250; this mechanism may be broadly applicable to type II cysteine desulfurase systems -cross talk at dimer interface, two monomers interacting and communicating -> half site mechanism

figure 5

HDX-MS data for peptide 88−100 under various experimental conditions. The data show deuterium incorporation plots for SufSapo (black circles), SufSper (red squares), and the SufS/ SufED74R complex (blue diamonds) -changes in protein dynamics at the active site are seen under numerous experimental conditions, but changes at the dimer interface are found only when one of the protein partners is in an activated or intermediate state (SufS with SufE); shows decrease in deuterium exchange when in persulfide form, but increase when when in complex with SufE -suggest that the interactions at the dimer interface are sensitive to complex formation by activated SufS/ SufE partners and may play a role in the proposed half-sites regulatory mechanism

figure 3

HDX-MS results for SufSapo and SufSper for 4 specific peptides (2 active site-255 and 356, 2 dimer interface-88,243); used to analyze conformation of persulfide intermediate of SufS, used to determine changes in backbone dynamics in SufS upon formation of the persulfide intermediate -found active site peptide with C364 has reduced deuterium uptake, same with the second active site peptide; consistent with changes predicted by structural changes in the static structures -a loss of conformational flexibility for regions at the dimer interface of protein upon persulfuration as well; Peptides 88−100 and 243−255 display a noticeable decrease in the rate of deuterium incorporation for SufSper, compared to that for SufSapo (the 88 peptide includes residues functioning in forming salt bridge at dimer interface, the other contains a flexible loop near B-hairpin motif involved in SufE binding) -suggest that SufE binding can cause changes at the active site of SufS, these two peptides have also been identified here as undergoing a change in dynamics upon formation of the C364 persulfide intermediate on SufS in the absence of SufE, must be a complementary role for SufE in active site remodeling of SufS to promote formation of persulfide intermediate

Estimating MW from SEC

Mr is the apparent MW of a protein from gel filtration chromatography; Hav is the partition coefficient that describes how a protein eluted from a column; Ve is the elution volume, Vt is the geometric volume; can plot log MW (x) versus Kav (y) to get a standard curve, solve for Kav for unknown MW of isolated protein using its elution volume in SEC, get MW of protein; Kav=Ve-Vo/Vt-Vo (Vo=all space not filled by stationary phase, Vt can be calculated by pi(r)^2(length of column); Kav will always be the same for a protein in any column -more approx than mass spec

why is NaCl used in the buffer for SEC experiment

Size exclusion chromatography should separate proteins only according to their hydrodynamic radius (size). However, the dextran and agarose components of size exclusion chromatography resins contain residual amounts of ionic groups; proteins having the opposite charge to the resin might bind due to ionic interactions while proteins with the same charge as the resin are subjected to ion repulsion and will elute earlier than expected; pH must also be maintained: cross-linking agents used in the manufacture of resins may introduce hydrophobic sites adding to range of modes of interactions between the resin and protein, these undesirable interactions may be decreased by adjusting pH

main findings from dunkle paper

Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS -SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE; reported five X-ray crystal structures of SufS including a new structure of SufS containing an inward-facing persulfide intermediate on C364; structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved β-hairpin structure plays a role in mediating interactions with SufE -> dimer interface interactions provide a way for two monomers to communicate between their active sites to shift from persulfide action of SufS and transferring to SufE acceptor protein -confirm a functional role for the dimer interface in mediating long-range conformational changes that affect the orientation of the persulfide intermediate and interactions with SufE.

UV absorbance of protein

Tyr and trp seen at 280 nm

what is the setup for liquid chromatography

a column filled with beads (carbohydrate based polymer that has been functionalized) called the stationary phase; next feature is the void volume (all nooks and cavities not filled by the solid supported/beads/this is the column); buffers flow onto the column called mobile phases, need to flow two under column with a value that allows us to mix the buffers, so we can create a gradient specific to the type of chromatography (pH, ionic strength, imidazole (metal chromatography); need to follow when proteins flow out the column -> use UV-vis (look at absorbance at 280 nm to look for proteins, proteins have tryptophans and tyrosines that absorb here); can also use a sophisticated UV-vis to look at visible light range (420 nm) specific for proteins that have PLP (5 prime pyridoxal cofactor); lastly a fraction collector is needed, maybe do manually or have a automated fraction collector -all together what do we have? FPLC (fine performance liquid chromatography)

darwins finches

a structural observation; darwin observed beak shape differences and recognized a correlation with food structure; early 20th century chemists could not see molecules; naturalists;

so how do we do this discontinuous assay

alanine is formed in rxn tube, take time stamps/samples/aliquots and quench them; add 5% Trichloroacetic acid (TCA) -> denature SufE and SufS so reaction stops, adjust rxn time point/inc volume, adjust pH to 9 with buffer, add in NDA and CN- (facilitate derivatization with NDA), take quenched aliquots and put into plate, fluorescence can then be read (more fluorescence, more alanine made)

What method is commonly used for protein purification

liquid chromatography

why do we have to do this discontinuously?

bc we do not have a continuous method to read how much alanine is being produced as the reaction progressed, have to do a discontinuous method to read reaction progress with NDA

how can we use enzyme kinetics to determine inhibitor mechanisms?

competitive, uncompetitive, and mixed; useful for tamiflu-> competitive inhibitor (binds to active site and blocks enzyme function) of neuraminidase (flu cleaves cyalic acid residues off of glycoproteins on surface of cell so it can leave the cell and move on)

two different kinds of assays

continuous versus discontinuous; continuous: could be a spectrophotometric assay, reaction may lead to a color change so we allow rxn to proceed and observe absorbance in real time (change in abs is proportional to the amt of product formed); discontinuous: haved to do this for cys assay because we need a quenching step (cannot analyze amt of product in real time), run reaction then have a quenching step to end reaction, the read out step

planes of crystals which scatter x-rays

crystal is made up of unit cells, translate unit cells into each direction x,y,z we can reproduce entire structure of crystal from unit cell (fundamental unit); divide unit cell into integer number of units we will get these planes bragg described (planes are parallel but intersect a common axis one time per unit cell; divide that axis by 1); adding additional planes, we divide that axis by 2, 3, etc; plane language (h, k, l); h derive from partitioning a, k derive from partitioning b, l derive from partitioning b

what else do we need in an x-ray diffraction experiment besides the x-ray

crystal, detector to collect diffraction, computer algorithms to process data and reconstruct 3D coordinates of proteins; need crystal for signal amplification (each crystal has trillions of individuals protein molecules in it, arrayed in specific arrangements next to each other; signal amplification effect instead of just getting diffraction patterns from a single peptide, proteins arrayed in space to get signal amp; technology does not have sensitive enough detectors or intense enough x-rays to measure x-ray diffraction from a single molecule of protein we could but we don't have this; why do we need computers: we do not have an x-ray lens (visible light has different speed in air vs glass so glass refracts light waves create glass lenses that shape light and take light that is refracted from an object and refocus to a spot and reconstruct the images; x-ray does not have a lens though, 1.5 Angstrom wavelength of radiation between C-C (a material to structure the shape of an x-ray so no lens)

table 1 that statistically describes the quality of the data for x-ray crystallography

data collection and processing: includes resolution (spacing on the high end of h,k,l planes that they were able to resolve is at 2.0A; on the low end of quality data the spacing might have been 2.7A; in refinement section: R/Rfree number tell us how well the model (x,y,z coordinates of the atoms and the b factor occupancy) explain the underlying data, lower R value is better (lower means be agreement between model and measured diffraction data)

once we make an electron density map, does it give us the structure of the molecule?

depends on the quality of map; if in range of 2-3 angstroms, gives us overall shape of AA in protein but doesnt show us where each atom lies; resolution affects the quality of electron density map (low resolution such as 6 A only show us tubes of density with little side chain information; up to 1.1 angstrom resolution we get much more info on placement of atoms); electron density model gives us ball and stick model but for more medium resolution data there is a level of interpretation that has to occur to build a model from the map

what's another issue with bradford assay

different protein standards give different standard curves; main thing that changes the proteins affect in binding to coomassie dye has to do with the # of Arg, Lys (basic residues), unusual amt of basic residues can skew technique, need to choose standard that has similar numbers of residue as protein of interest; A595 signal depends on [protein] but can vary by protein identity

why do we need x-ray crystallography

diffraction limit (use radiator to probe) tells us that the matter we are solving cannot be much smaller than the wavelength of radiation we are using. ex: if we are using 400 nm (visible light), tells us we can observe things spaced 200nm apart, if the are smaller than that we won't be able to solve them at 400 nm; studying protein structures: have c-c bonds 1.5A, need wavelengths corresponding to less than an angstrom-> use hard x rays that have wavelengths 0.7-1.5 Angstroms

figure 5 dunkle

examined interactions between CsdA/E (homologous to SufS and E); shows interactions at dimer interface, shows that the active site E cannot reach the persulfide on A (indicating another confirmation is necessary for transfer), found the beta hairpin structure blocked the active site -> beta hairpin may regulate when transpersulfuration can occur -R88 interactions at the dimer interface may regular movement of beta hairpin and therefore the transpersulfuration -movement of beta hairpin is necessary for transpersulfuration/reveals an interaction network, similar to that in SufS, exists, which may modulate β-hairpin dynamics

what are assays used for

follow enzymatic rxns

what did pauling and corey do? (in addition to geometric observations)

had to deduce structure of alpha helices and beta sheets using observations without known structure; observed that peptide background could participate in hydrogen bonding interactions (dipole-dipole interaction, varying levels of energy depending on geometry of bond); in early 1950s, observations from x-ray crystallography of dipeptides and H-bonding interactions he came up with alpha-helices: right handed helix, 12 aa avg length, pro, gly, rarely occur

what is the key issue in getting a multiple turnover rxn (high throughput)

have to be able to reduce SufE persulfide back to reduced for for multiple turnovers -> cant do this by reconstituting rxn with sufBCD (reduced creating iron sulfur clusters) bc impractical, instead use a chemical reductant TCEP; this rapidly reduce SufE into sulfhydrl form so we can continue rxn as multiple turnover rxn

how to determine protein structure

high resolution techniques that can be done in the solution phase: NMR, single particle reconstructions by cryo-EM (can lead to molecular models); low resolution techniques in solution: SEC (determine quat), native PAGE (quat structure), ultracentrifugation (quat structure), and circular dichroism (determines amounts of alpha helices, beta sheets, secondary structure; High resolution in crystal: X-ray crystallography, electron crystallography

relationship between Fhkl and e- density

if we have a structure of the molecules in a unit cell, we can calculate what bragg peaks we expect to see; we can also use the peaks/diffractions on detector and then calculate what is the row/electron density that explains the amplitudes or intensities, phases of the observed diffracted x-rays; intensity (look at diffracted spots, some are higher and some are lower; tells us distribution of atoms in h,k,l plane of that diffracted x-ray) and phase (information is lost, cannot collect this info on detector, have to use computers to recollect it through molecular replacement; sometimes not possible to recover this way) are important

buffers for IEX (buffer controls what happens in experiment/pH/when protein will elute)

in cation exchange, acetate is a good buffer (acetate has same charge as the column so it will buffer between negatively charged and neutral so it won't bind to the column, won't interfere with protein binding bc of COOH); in an anionic exchange, Tris is a good buffer (has an amino groups so it goes between being neutral and negative and will not interfere with negatively charged proteins attaching to column bc it has a similar charge to the column, proteins will interact with mobile phase but wont bind to column)

how do we determine what kind of inhibitor we have using kinetic assays?

in each steady state assay, use a set concentration of inhibitor, perform multiple experiments, increasing amt of inhibitor in each; end up with different curves (plot at straight lines using lineweaver burke plots/double reciprocal instead of hyperbola)

what are the four common types of liquid chromatography

ion exchange (cation and anion subtypes; take advantage of the fact proteins have different amount of charge on exterior; IEX), size exclusion (take advantage of proteins of different sizes), affinity (different than the rest, not looking at physical aspect of protein; looking at specific chemistry of protein that others do not have, IMAC approach (we do this; stationary phase where nickel ions are immobilized, proteins rich in HIs will bind tot the resin with high affinity and others will not), and hydrophobic interaction chromatography (taking advantage of proteins different hydrophobicity on exterior, determine how tightly or loosely they bind to stationary phase)

why do we need to purify a protein a interest

isolate a protein from other proteins present in a cells lysate (complex mixture); SufS from other proteins in E.coli lysate; we used SEC

table 1

kinetic parameters for SufS wt and variants; 92 variant has most effect on kcat; All three variants exhibit increases of ∼2-fold in Km, effects result in 2−3-fold decreases in their kcat/KCys values relative to the wild-type parameters; found 6-fold decrease in 96 and 250 positions when adding SufE for Km, variants exhibit 6−7-fold decreases in their kcat/KSufE values relative to those determined with the wild-type enzyme -suggests residues at E96 and E250 play a role in the interaction between SufS and SufE at the dimer interface as substitution does not affect catalysis (i.e., kcat) or interactions with cysteine -The 6- or 7-fold increase in KSufE values correlates well with the magnitude of the change seen in the Kd values for SufE and activated SufEalk/D74R; interactions at the dimer interface appear to be necessary for optimal interactions between SufS and SufE

Size exclusion Chromatography

large proteins come out earlier because they don't get stuck in the semi porous matrix; isocratic elution means we used a constant mobile phase (one buffer), continuous flow rate of a buffer; larger migrate faster, path of smaller proteins takes much longer to go through column; results in chromatogram

Beer-Lambert Law

law stating that intensity of color change is directly proportional to the concentration of an analyte in a solution

what is Vmax

maximum velocity we can achieve is we saturate the enzyme with substrate; hyperbola curve, at the far right of the graph even with increasing amounts of enzyme we will eventually hit a maximum/same initial velocity; Vmax=kcat[E]total (we have added so much substrate so every mole of enzyme is bound to subs), useful bc we can rearrange and find kcat (activity of enzyme/turnover number); makes it easier to compare across journals and experimental conditions, same kcat always if only enzyme amount is changing

so what are the steps to obtaining a structure of a protein and solving a x-ray crystal

obtain protein crystals, perform diffraction exp, calculate electron density maps -we used the Rigaku Synergy DW X-ray diffractometer

why can is be complicated developing assays to follow enzymatic rxns?

often times the substrate looks similar to the product (cys, alanine)

mass spectroscopy experiment

performed trypsin digestion first by using purified SufS, ultrapure water, 2x trypsin-ultra reaction buffer, and trypsin-ultra enzyme (incubate overnight); desalting of trypsinized peptides: added TFA to digestion rxn; aspirate wetting solution into tip and then aspirate equilibration solution (both containing 0.1% TFA), aspirate sample 7 cycles for maximum binding of the mixture, aspirate wash solution then elution solution; maldi target prep: prepare DHB (2,5 dihydroxybenzoic acid) matrix using 20mg/ml in 30:70 v/v acetonitrile:0.1% TFA in water, mix eluted peptides to DHB matrix and apply to maldi target, allow peptide matrix to evap then insert target into mass spec

isoelectric point

point at which a protein has exactly the same amount of positively charged AA as negatively charged AA; say a protein has mostly basic residues at pH 7, we increase pH so they lose hydrogen now protein is becoming more neutral; we can do the experiment at different pH mobile phases and manipulate the charge on the surface of the protein because of this isoelectric point

how were samples prepared for alanine standard curve for cys desulfurase assay

prepared 0uM, 5uM, 10uM, 20uM samples; ultrapure H2O, 10X MOPS, Ala 200uM

diffraction of x-rays by matter

primarily interested in elastic scattered (incident x-ray is scattered by electron cloud of an atom, emergent x-ray has same wavelength as ray initially contacting atom/no absorbed or lost energy); how did bragg describe the way crystals diffract: imagine we have 2 atoms, 2 x-rays are scattered by each of these two atoms (x-rays are parallel, distance between atoms and angle that describes interaction of atoms with x-ray called theta), draw a from extending from xray 1 to x-ray 2, we observe that x ray 2 to scatter off of atom 2 has to travel slightly farther than atom 1 does (length l), use geometry to determine l (hyp is the distance between two atoms); we find x-ray 2 travels travels a longer distance than x-ray 1 by 2l (equal to integer number of wavelength) -> x-ray 2 will be in phase with 1 when two wavefronts meet each other (2dsin(theta)=nd; n=integer number of wavelength); only when we have this constructive interference between waves between atoms in a crystal will we get a diffraction pattern on detector; bragg's law! - think of this as two planes of atoms in a crystal instead of just two atoms: when crystal is rotated so that theta is set up and brad condition can be met (distance between the planes = nd, integer number of wavelengths) diffraction will occur

protein structures

primary (amino acid sequence), secondary (alpha helix-beta sheet, random coils), tertiary (folding pattern, how do alpha and beta segments come together to form 3D structure), quaternary (multiple polypeptides interacting together/heterodimer/homohexamer)

SEC experiment

prior to SEC: protein is concentrated (purified by IMAC), 10% glycerol added as a cryoprotectant to prevent damage of cells during freezing/stored at -20; further purified protein by removing aggregates from sample, eluting before SufS (larger molecules); SEC done on FPLC to purity protein and figure out quaternary structure; protein first incubated at 37 degrees C to disrupt weakly interacted oligomers, sample loop mixed with MS buffer (NaCl, MOPS), dialysis tubing rinsed with water, S200 10/300 column used, needle kept in so air could not get in, protein loaded onto sample loop; A280/A420 FPLC curves used to identify major peaks containing SufS (Void at 8ml); SufS fractions added to 10000 MW cut off centrifugal concentration device to concentrate SufS (Pall Macroprep) and centrig=fuged for 1 hour at 4000 RPM, glycerol added again before flash freeze and storage

Anfinsen experiment

protein amino acid sequence dictates amino acid structure (thermodynamic hypothesis: folded structure is the thermodynamic minimum structure that can be adopted); Rnase experiment, lowest deltaG adopted

how do these methods actually get used?

protein data bank (rcsb.org); almost 90% done by x ray crystallography; x-ray crystallography is most challenging (need crystals that are well-ordered), but generates most atomic structures of proteins that we have available

how do we know proteins have discrete structures?

proteins are polymers of AA- typically no discrete structure), found in the 20th century that is not the case for proteins (Bragg crystals diffract in X-rays, lead to Xray crystallography and determining molecular structure); thought proteins did not have 3D structure bc their crystals did not diffract x rays(bc most inorganic structures, solvent is not necessary for maintaining the order of an inorganic crystals; we knew proteins could crystallize (pepsin) but when those crystals were handled the same way as their inorganic counterparts were (not maining in aqueous solvated state) then they could not diffract (disordered); bernal and hodgkin found that protein crystals had to be solvated in order to maintain their 3D structures (ordered structures, crystals can diffract like other molecules); corey and pauling found a way to find the 3D structure of proteins: solve x ray crystal structures of dipeptides (key information found, aided in determination of protein secondary structures); in 1958, kendrew solved first 3D structure of protein by x ray crystallography (myoglobin), showed what proteins look like in 3D

In our case.. (IMAC experiment)

proteins engineered with 6-His at the C terminus of our protein (can be put on N terminus); translationally fused to protein; immobilized metal affinity chromatography; Ni2 (Ni-NTA) - what we used, or Cobalt can be used for resin

affinity chromatography

purifying His tag of SufS using IMAC; apply mixture of proteins to IMAC resin, allow it to flow through column, proteins that don't have his tag will elute in wash steps (contaminate proteins that are weakly bound will come off resin); then apply solution of ligand (imidazole mimic histidine and interact with column, causes desired protein to elute from resin in elition fragments); chelating resin: two his residues next to each other can displace water and correlate to the Ni ion - strong interaction

michaelis menten is used to

quantitate activity of enzymes; amenable to lab investigates (single lone input, substrate concentrate, product amt/concentration as function of time -> can be used to monitor activity of enzyme using MM expression

so what is a peptide?

repeating unit of N-Calpha and then another carbon (hundreds of units); amide bond in backbone of protein is not only a double bond between C and O, single bond between N and C but that there is resonance occurring; the no rotation around peptide bond, found there were two different planes between C alphas (linus, pauling-> found in crystal structures of dipeptides, found different lengths between different N-C bonds); rotation around C alpha bonds to C and N: C apha looking at nitrogen rotation is a positive number/phi dihedral angle, C alpha looking at carbon carbonyl rotation is also clockwise and is positive/phi (this is important because it limits protein structure/only two degrees of freedom in background, constraints what arrangements of polypeptide background is possible (only some phi and psi values possible)

2nd form of braggs law

series of h,k,l planes positioned with a certain theta angle relative to incident x-rays so that spacing between planes is equal to an integer number of wavelengths, we see a bragg peak/diffraction peak; goal of x-ray crystallography is to rotate pass through all theta angles to eventually bring combination of h,k,l into x-ray reading

figure 1 kim

showing SufS structure in E.coli, C364 and K226-PLP internal aldimine shown as spheres; showing the mechanism of SufS: first half as the persulfide formation, cystine desulfurase action, then giving sulfur to SufE acceptor protein (to Cys51) to relieve SufS protein -showing basic mechanism/most important aspects of SufS

figure 3 dunkle

shows electron density maps; confirmed the presence of persulfide; found that the active site cysteine can form a conformation facing towards PLP and away; suggests different conformations of the active site where SufS accepts sulfur atom and when it presents it to SufE -Both positions must occur during the catalytic cycle as C364 accepts the persulfide (positioned in) and then presents it to SufE (positioned out) -the inward-facing conformation of C364 is an earlier intermediate in the SufS reaction, after the desulfuration of L-cysteine has occurred but prior to full transition to the transpersulfuration step -two separate conformations at active site may be dictated by dimer interface interactions, and this also can communicate to transitioning into the transpersulfur reactions of cys desulfurase

figure 4

shows electrostatic interactions of R92 in SufS (involving active site peptides investigated by HDX-MS); shows structure of SufSapo dimer and the superposition of peptides under investigation (R92 and E96, E250), when bound to external aldimine analogue; Ribbon structure of a SufS monomer with the β-hairpin and peptide 243−255 highlighted with the PLP cofactor shown as a space-filling molecule -E96 and R92 form salt bridge at dimer interface, shows the peptide containing residues 243−255 forms a flexible loop region between the main β-sheet and a β-hairpin motif at the dimer interface; when SufS is bound to an external aldimine analogue, R92 shifts away from its interaction with E96 and forms a new interaction with E250 providing a link between persulfide induced changes and the dimer interface; The decrease in the rate of deuterium incorporation in these two peptides upon persulfuration of SufS suggests a conformational change occurs in this region but because no differences were observed when SufE added, may specifically play a role in the half-sites regulation -interface region (250) perturbed upon persulfide formation -changes at the dimer interface are responsible for communicating the persulfide status of a SufS monomer through modulation of the hairpin structure

figure 2

shows possible dimer face interactions of SufS wt and mut (at dimer face interactions) and how these interactions shift the overall positioning of the dimer molecule as well as specific changes occuring at the active site -main point is that dimers communicate and there is crosstalk between monomers; consistent with data suggesting half-site mechanism of sufS shifting between the active sites of the monomers; interactions at dimer interface control active site conformations -persulfide state of an active site could be communicated to the adjacent monomer via connections between the persulfide intermediate and S254 amide transmitted through the dimer interface

figure 1

shows the differences between type 1 and type 2 cysteine desulfurases: flexible loop found on type 1 cys desulfurases (mechanism for transferring sulfur, accessory protein helps orient), type 2 has a more rigid loop with active site residing on it (more complicated transfer, two types have two different mechanisms) and specific acceptor proteins are required; type 2 also has an additional beta hairpin motif (the hairpin from one monomer reaches across the dimer interface to interact with the active site on the adjacent monomer) -showing overall differences between the two, clue into how type 2 cysteine desulfurase must function with an accessory protein so interaction with it must be under control and specific

Figure 2

shows the peptide digested map generated by MS/MS of nonreduced SufS and reduced SufS using TCEP (gets rid of persulfide); Cysteine active site peptide can only be seen after applying reductant -used to identify the location of the generated persulfated intermediate; no coverage was seen for C364 active site cystine in nonreduced persulfide state of SufS, while the other three cysteine residues were found in their native state; then looked at sequencing of reduced persulfide to see if the persulfide at location had interfered with sequencing -> it did, suggests that persulfide had formed and was present before reduction; consistent with a species containing one persulfide moiety on C364.

scheme 1

shows the two separate functions of SufS cys desulfurase: desulfurase and transpersulferase actions; mechanistic challenge in shifting between forming persulfide intermediate and then making it available for transfer to SufE -main point is to show two activities of SufS/must be a way for the enzyme to shift between activities of these two separate functions

how are cysteine and alanine similar/different

similar: amino group, carboxylic acid group, MW similarity, polar molecules (dissolve in solvents) dissimilar: sulfhydryl functional group on cys - must set up an assay that will distinguish between cys and alanine (based on SH group)

crystal growth

some concentration at which a protein is soluble in water (but upper limit to solubility, conc where it is no longer soluble); protein may be supersaturated right above solubility range (thermodynamically system is not at equilibrium, protein wants to come out of solution but kinetically it has not yet; key to producing good crystals large and well order is to slowly transition protein to supersat point, give it some time there for molecules to come together and form a nucleus to template out into a crystal (take protein too rapidly from soluble to insoluble region, you get aggregated solid material -> not thermodynamic minimum but driven by kinetics processes so it is not a regular crystal and wont diffract); vapor diffusion allows us to slowly reach thermodynamic point (precipitant is a compound that allows you to alter solubility of protein, such a PEG, salts, or low ME alcohols): at low precipitant concentration, protein is more soluble, less precipitant, less soluble; in vapor diffusion exp: mix protein with precipitating agent, starting at a specific concentration (allow water vapor to diffuse out of a drop, allows drop to sink so protein and precipitant will become more concentrated a function of time), drive protein precip mix into supersaturated region gently so there is time for crystal to form, concentration of protein then will drop and experiment will end in a new position where amt of soluble protein for growing the crystal runs out

IEX cationic exchange media

strongly acidic may have an SO3 groups, weakly acidic will use carboxylic; for anionic exchange media, use N-(4)-CH3 for strongly basic, and other amino groups for weakly basic

why perform enzyme kinetic measurements? what are the practical reasons?

to compare enzymes quantitatively: wt versus mutant - kim et al., enzyme from one species vs another organism (biotech purposes, which enzyme works best); determine mechanisms of inhibits (many drugs are enzyme inhibitors), to determine the mechanisms of bisubstrate rxns (random versus ordered rxns)

figure 6

structural representation of the CsdA/CsdE complex; the area in the red square contains dimer interface residues corresponding to those described with SufS; differences in missing electron density between the CsdA/CsdE structures are labeled and correspond to those described in the text; structure of alternate conformations and interaction partners of R88 in CsdA -comparing CsdA/E to SufS/E dimer interface reactions for similarities, found a connection between the dimer interface and region adjacent to beta hairpin/active site area; CsdA/CsdE complex reveals an alternate conformation for R88 (interaction with E92′ of the adjacent monomer, similar to the structures of SufS, in the alternate conformation R88 interacts with A271), A271 is located at the C-terminal end of the β-hairpin, in contrast to E250 in SufS (n-terminal) but both structures highlight a connection between the dimer interface and a region adjacent to the β-hairpin -structure also provides evidence of the role of this interaction in a half-sites model due to a lack of symmetry: no density show near β-hairpin of CsdA or persulfide acceptor loop of CsdE suggesting they are flexible when the two are in complex; R88 can be seen with E92 when only they are interacting (no acceptor protein); This suggests that changes in orientation of the dimer interface arginine residue can affect the flexibility of key portions of both the desulfurase and the accessory transport protein

mass spectrometry

tandem MS is used to reveal the AA sequence of peptide constituents of protein (AA determines 3D structure), used to unambiguously verify the protein is SufS; protein sample is digested with trypsin protease (cleaves amide bond of the peptide backbone following positively charged residues like Arg and Lys; we used NEB trypsin-ultra) breaking the protein into peptide fragments of 1000-3000 Da; this peptide fragment is ionized and mass is measured then directed to collision cell containing an inert gas (He); KE resulting from the collisions of the peptide with has causes cleavage of covalent bonds in the backbone of peptide; then compare m/z of the precursor peptide to the collection of fragment peptides (each smaller than precursor by an integer number amino acids) to deduce precursor sequence; we used MALDI matrix-assisted laser desorption ionization

quantitative amino acid alaysis

technique used to determine the amount of commonly occurring amino acids occurring in tissue or purified protein; extract protein from tissue, treat with 6M HCl (hydrolyze peptide bonds in protein, reducing to constituents amino acids), apply liquid chromatography in ion exchange column (separate AA), derive AA using ninhydrin (reacts with amino group) -> detect AA on chromatogram as they flow off of a column -> get a chromatogram of abs versus retention time (each AA will have its own retention time), measure absorbance at 570nm for primary amino peaks (for primary amine peaks, 20 peaks found), if we want to monitor prolenes measure at 440 nm (secondary amine); integrate peaks to get amount of AA present

what are some things to consider when setting up an enzyme assay?

time resolution, sensitivity, safety (radioisotope usage), and throughput (need high throughput); hard to develop assays because substrate and product are so similar

ion exchange chrom

two kinds: cation or anion exchange (named based on what binds to it, stationary phase is negatively charged is a cation exchange bc proteins with positively charged amino acids will bind; proteins rich in neg charges we use anion exchange); pos charge ions in mobile phase interact with column, when protein binds it displaces the mobile phase; for both techniques we run FPLC and get a plot of abs at 280 nm versus Vo or time (if we run at 1ml/1min then both will be the same); ex: in cationic exchange, negatively charged will be repelled by stationary phase and elute very early//basic residues will bind to column;we select for proteins that bind to the solemn weakly vs strongly, we mix buffer A and B to create a gradient of sodium chloride (step or linear gradient; start at low NaCl and add as column runs, increasing ionic strength of solution so proteins that have strongest affinity to column will elute last)

SDS-PAGE analysis experiment

used coomassie blue stain to die protein - binds well to protein and turns it blue so we can visualize

protein quantification experiment

used to determine concentration of our purified protein (bradford and nanodrop technology) -nano drop method: first determine extinction coefficient by measuring number of tyrosine and tryptophan in protein, blank nanodrop with MS buffer, invert tubes to ensure good mixing and apply to nanodrop pedestal, instrument displays A280 value and can be used in beer lambert's law to figure out concentration of protein (done twice) -bradford assay: determining protein concentration using a bovine standard curve; bovine serum albumin stock was used (similar pos/neg amino acids as our protein) to prepare curve (measured A595 for a series with diff concentrations (NaCl, bradford reagent, and BSA), prepared 1:100(1uL, 19.9uL of NaCl) and 1:200 (2uL and 19.8 uL NaCl) dilution protein sample in 0.15M NaCl for total volume of 200 microL, used samples of 100 microL protein; blanked GE ultrospec then measure abs at 595 (find concentration using excel, multiply by 1000 and 2000 to get amt in grams; MW of sufs 44434 g/mol to convert ug/mL to uM)

protein purification using IMAC chromatography experimental methods

used to isolate and purify recombinant EcSufS enzyme variant; steps: cell lysis, IMAC chromatography, SDS-PAGE analysis (samples saved throughout steps to see purity of final sample); lysed cells by sonication using PMSF, DNasel (made viscous), lysozyme (damaged cell wall); used ultrasonic dismembrator to sonicate cells (total 10 mins sonication), took a sample for whole cell lysate -> microcentrifuge then separate supernatant and pellet (lysis load, lysis pellet); centrifuged cells in F15 rotor to pellet the cellular debris, keep supernatant which contained soluble protein -now IMAC: used a Ni2+ imac resin in binding buffer, cell lysate added to it to obtain flow through sample (6-His tag interacts with Ni2+ on resin, stays on column longer while other cell contents leave in earlier samples using wash buffer), collect 4 wash samples, 4 elution samples that contain protein - higher concentration of imidazole in elution buffer to allow protein to flow through column

A280

using beer lambert law (c=A/b(epsilon); epsilon280 = (#Trp (5500 1/M cm)) + (#Tyr (1490 1/M cm)); may not work well if we have a molecule that also absorbs at 280nm that may interfere and we cannot get an accurate absorption value

cysteine desulfurase assay experiment

we measure the progress of the reaction by quantitating the alanine product; NDA method: will derivatize and detect amino acid, is useful in cys desulfurase assay bc the reaction with cys does not produce a fluorescent product but it does with alanine; assay components: ultrapure H2O, 10X (500mM) MOPS, Cys 500 uM, TCEP 500mM, Ala 200 uM, EcSufE wt, EcSufS wt, EcSufS mut, detection buffer (0.1 mM NDA, 1mM KCN, 100 mM sodium borate pH 9), 96 well plating containing 5uL 10% TCA in wells; TCA ends rxn (quenching step - account for discontinuous, have to take timed samples to measure progress; used TCEP to reduce SufE so reaction can continue as time progresses), NDA/KCN used in reaction with NDA with alanine product to provide derivative of NDA (fluorescence measured) -first made alanine standard curve using different nmolar amount of alanine, measuring fluorescence -> use equation later to determine nm of alanine in cystine assays; before SufS was added, we took a sample at 0mins and quenched, then added SufS to initiate rxn and took samples at 3,6,9 mins then quenched samples (for both wt and mut); detection agent was then applied to each, wait 20 mins, Biotek Synergy plate reader run with an excitation wavelength of 390nm, emission of 440 nm -plot nmol alanine (found from fluorescence and standard curve) versus time and perform linear regression for both wt and mutant, the slope is Vo (nmol/min) for the reaction at a given concentration of Cystine (v=k[ES])

what is NDA?

with present of CN-, reacts with amines to create a derivative which is fluorescent, giving an excitation peaks at 420nm and an emission peak at 490nm; better than ninhydrin bc these adducts are fluorescent so they are more sensitive detection and they are more stable

bradford assay

works for 1-10 micrograms of protein; coomassie dye binds to proteins (color change), absorption value depends on proteins ability to bind to the dye; dye either has cat, neutral, or anion form -> protein binding causes it to be in anionic form, absorbance at 595 nm; make a standard curve with known amounts of protein (account for spectrophotometer mistakes) using proteins that are easily obtainable (bovine); use our protein, see where it absorbs at 595nm to determine concentration

determining protein structure by

x-ray crystallography; allowed chemists to observe the structure of molecules; been around for 100+ years (2014); diffraction image from Rosalind for DNA was taken by fiber x-ray diffraction in 52, myoglobin and lysozyme structures determine in 50-60s, synchrotron built in 70 (intense source of x-rays, tunable for wavelength), solved ribosome structure, viral structures, etc (gives us spatial information of molecules); free electron laser technology has given us maintain spatial resolution and capture time resolved images of proteins undergoing reactions

braggs description of planes in a crystal acting as mirrors to scatter x-rays is a useful analogy

x-ray interacts with electron cloud - scattering occurs in all directions; significance of the spacing between the atoms is the d'spacing from these spherical waves that are produced is the spacing that gives constructive interference