Exam 1 MCDB 310 Professor JK Nandakumar

2D electrophoresis

2D electrophoresis combines SDS-Page in the vertical direction and running that also with isoelectric focusing in the horizontal direction! 1. Isoelectric focus 2. Put this in the SDS-Page and the proteins now run down vertically helps with 100s or 1000s of proteins in a cell needing to be resolved from each other and analyzed separately - Proteomics

Discussion ramachandran plot

where the alpha and beta likely to form!

Kinetics (NOT thermodynamics)

the speed of reactions... reaction rates! Depends on the ACTIVATION ENERGY barrier (dG double dagger), and is INDEPENDENT of dG!!!!! CATALYSTS lower Ea without affecting dG. - in the cell, called ENZYMES

Lecture 5 Chapter 4 continued Globular proteins: properties/structure micelles and diff. two sites domain question from quiz

- Hydrophobic interior CORE and Polar exterior on the suface (similar to Micelles!) key difference from micelles: It is all ONE molecule, not a bunch of them together. - Combination of Structural motifs -- secondary structures group together to form motif, no function on its own but important for function/properties of domain! - Niches and crevices where cofactors bind - have an ACTIVE SITE and a substrate BINDING SITE - Larger polypeptides have 2 or more stable globular units called DOMAINS (functional units where each has its own function, but is part of the SAME primary structure protein!) "independent folding unit" QUIZ: Domain= polypeptide sequences that can fold into a 3D fold with tertiary structure and perform particular functions even when present as a subset of the complete amino acid sequence of a protein

Amino acids properties 1. all aminos... (connectivity and shape) 2. All EXCEPT proline have a... 3. All amino acids EXCEPT glycine are ____

1. ALL amino acids have 4 substituents and is tetrahedral - carboxyl group, amino group, alpha carbon, hydrogen, and side chain 2. All amino acids have a basic PRIMARY amino group BESIDES proline (considered a secondary amino group) 3. All but glycine are Chiral! Chapter 3 slide 19-20 examples of amino derivatives/modifications

Ionization of amino acids simplest form

1. Acidic pH!: the amino acid is positively charged with carboxylic and amino group protonated - pK1 2. Neutral: Zwitterion forms as the carboxylic acid loses a proton making a positive/negative species 3. Basic pH: amino group becomes neutral and so the amino acid becomes overall net negative

Biochemical reactions 1. group transfer reactions 2. Oxidation and reduction reactions

1. An electrophile and good leaving group reacts with a nucelophile to exchange A-X + Y -> A-Y + X - leaving group X is good when large and stable with negative charge (acidic) - Peptide bonds form through making a good leaving group (O-tRNA) and then the N attacks and kicks it off. 2. Oxidation/reduction reactions Oxidation: Loss of electrons (lose hydrogens, gain oxygens) Reduction: Gain electrons (gain hydrogen, lose oxygens) ex: NAD+ and FAD, glucose oxidiezed to CO2

How do we know how much protein we have overall? (light) how does it work, what absorbs light? Equation explain the graph

1. Aromatic amino acids (Phenylalanine, tyrosine, Tryptophan) absorb light in the UV region 2. proteins typically have UV absorbance maxima around 275-280 nm 3. W and Y are strongest chromophores (release strongest light) ****Concentration can be determined by UV-visible spectrophotometry using BEERS LAW A = ε c L ... Absorbance epsilon= molar absorptivity constant L = path length, length light passes through sample c = concentration (M) KEY***** For epsilon, you add up all the Y F and W in the molecule and then multiply those numbers by their epsilon and add them together for the total! - Absorbance comes from graph and wavelength, find epsilon and you find concentration READING THE GRAPH: Tryptophan is much higher at A280 which means its epsilon value is greater

5 ionizable side chains for titrations are... Titration for D and E (aspartate and glutamate)

1. Lysine (K) 2. Histidine (H) 3. Arginine (R) 4. Glutamate (E) 5. Aspartate (D) HERE: titration curve for Aspartate and Glutamate Note: pK1 < pKr < pK2, but pK1 and pKr are close and pK2 is far away

SDS-Page can be used to check purity and estimate molecular weight...

1. SDS-Page can be used with coomassie to check if the protein is pure or if other things are present. 2. SDS-Page can be used to estimate the MW, not accurate. - run with known marker and you plot, then plug on graph and extrapolate by using its relative migration distance.

QUESTIONs (MCAT examples)

1. What is the pH given 5 mM HNO3? - no calc, you know -log (1 x 10^-3) = 3 - log (10 x 10^-3) = 2 must be between 2 and 3! 2. What is the pH of a 20 mM acid and 200 mM CB with pKa of 4.76? - no calc, more base than acid so you know it will be greater than 4.76! log 10 = 1, ... 5.76 is answer

Most simple titration curve for an amino acid... - properties at zwitterion? buffers? How to calculate the pI of aminos WITHOUT ionizable side chains?

1. acidic pH, positive charge, add base and the carboxylic acid deprotonates, at pK1 it is pH=pKa of the carboxylic acid deprotonation 2. zwitterion forms and is the ONLY species present at pI (isoelectric or equivolence point) -Does not migrate in an electric field since is pulled both directions - LEAST soluble in water, the positive and negative charges stabilize the ion. 3. very basic pH, the positive amino group now will deprotonate and here the pK2 represents the point pH=pKa this means all amino acids can buffer at two ranges... some maybe more *******ALL amino acids the carboxylic acid to the next species is the pK1, and the species to the alpha-amino group is the pK2! the pI is the zwitterion! pI = pK1 + pK2 /2

Phi and Psi and Dihedral Angles, how it relates to ramachandran plot

1. some phi and psi combos are favorable due to H-bond/ionic interactions/etc. 2. Some are unfavorable due to steric or rejected charges 3. a ramachandran plot shows the distribution of phi/psi dihedral angles that are favorable or unfavorable in a protein

Lecture 1 Review Chapter 2 Water Molecule unique properties

65% of the body is WATER (not biomolecules) 1. bent molecule with tetrahedral geometry (2 localized lone pairs) 2. Hydrogen partial positive, oxygen partial negative

How do we measure the binding affinity of a complex?

= fraction of binding sites on protein bound by ligand ... degree of saturation of the protein θ = [occupied binding site] / [total binding sites] - may also be seen as Y on a graph's y=axis (upper case theta) 0 < θ <1 - less than 1 but greater than 0!

Thermodynamics ... Spontaneity and coupling

A reaction will be spontaneous if dG < 0 Equilibrium is when dG = 0 non-spontaneous when dG > 0 Couple a spontaneous rxn (ATP -> ADP) with non=spontaneous! - dG1 +dG2 = dG3 so dG3 can end up being spontaneous!

Isoelectric focusing electrophoresis

A sample of proteins which have a certain charge/acidity/basicity will be placed on a strip with immobilized pH gradient. The protein will stop moving when pH =pI (zwitterion, not pulled either direction in an electric field) Isoelectric focusing helped determine a proteins pI

GRE questions... lysine mutation ... most and least effctive

A. glutamate is most harmful... before you had a positive now negative... before it was lysine (+) forming ionic interactions which now cannot form/repel the DNA backbone B. Arginine: least, still negative and semi similar structure

Intrxn #4: Van Der Waals interactions

ANY TWO atoms or molecules have an attractive force or optimal distance away from one another regardless of charge or size. Usually between non-polar molecules. Adding thousands of small interactions make it very strong or important. Van der Waals Radius: the distance from one atom to another (which are non bonded!) is the diameter, so half that distance (the distance to the midway point between the two) is the VDW radius. cumulative effect CRUCIAL FOR: macromoelcules - an enzyme approaching a substrate... water cage around substrate and enzyme break and other bonds (ionic, VDW, polar, h-bonds) stabilize the 'recognition'

Diseases with misfolding

Aggregates and fibrils form nasty aggregates leading to diseases! Alzheimer- amyloid Beta protein tau Mad cow disease- Prion protein Parkinson's: alpha-synuclein

Naming peptides + functions in body:

Always start the AMINO end -> carboxylic end! functions: 1. hormones and pheromones (insulin/oxytocin) 2. Neuropeptides (substaince P... pain mediator) 3. Antibitotics (polymyxin B (for Gram -bacteria) and bacitracin (for Gram + bacteria)) 4. Protection, e.g., toxins-amanitin (mushrooms)-chlorotoxin (scorpions)

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B. Amphipathic

Type of compound: Amphipathic

BOTH hydrophobic and hydrophillic elements... makes it behave as polar and non-polar 1. polar/charged/ionic - polar: glucose, glycine, aspartate, lactate, lactate, glycerol 2. also non-polar uncharged - Nonpolar: wax example: Lipids and some amino acids

The limited conformational space between peptide bonds in proteins results in SECONDARY STRUCTURE: 1. Alpha-helix 2. Beta-sheets properties/definition what id donor/acceptor of h bond side chain orientation two types of comnfigurations and their prop! (anti/par)

Beta sheets: NOT a perfect sheet since the phi and psi planes are not completely flat (180). - the planarity of peptide bonds and tetrahedral geometry however does create a pleated sheet-like structure - H-BONDS between different amide (donor) and carbonyl (acceptor) groups in aa's - Side chains protrude the the sheet alternating up and down (helps separate phobic, charged, etc.) - There is both parallel and antiparallel orientation of two chains within a sheet! strand = 1 (almost never exists by itself) sheet= many together - Antiparallel: H-bonded strands run opposite directions resulting in STRONGER, CO-LINEAR H-bonds - Parallel: H-bonded strands run same direction, not as directional H-bonds = weaker

Lecture 1 Review Chapter 1 Biomolecules

Biomolecules are any molecules developed during the evolution of the cell! - ORGANIC compounds found in the cell Not all of a cell is made up of biomolecules, they can be macromolecules but do not need to be! ex: carbs, lipids, proteins, nucleic acids

SDS-Page vs Size exlusion

Both will separate a protein by SIZE however... -Size Exclusion: does it in protein's native state and FOLDED... also, you do it with the ENTIRE sample to separate the protein out/purify it - SDS-Page: unfolds and separates proteins in an inactive denatured form (small amounts used in the separation)

Abundant elements in cells

C (60%) H (11%) N (9%) O (6%) -- ^Make up biomolecules Sulfur: amino acid bonds Phosphorus: ATP Salts in cells (Na, Cl, etc.)

Lecture 4: cont Chapter 3 Separation by Charge: Ion Exchange chromotography

CATION EXCHANGER EXAMPLE (opp. for anion) 1. proteins lysed from the modified bacterial cell in previous steps now will be placed into a buffer. 2. The buffer is run through a column with Cation exchangers - Cation exchangers (column RESIN) are BEADS which are anionic and therefore attract the more + charged proteins - Greater positive charge means more affinity for the beads/resin - NOTE**** basic proteins (think NH3+) bind STRONGER in a cation exchanger than acidic since they are more positively charged! - Used for basic if positive, negative for acidic! 3. The protein then run through has the NEGATIVE and less positive proteins elute out since negative repels the beads, leaving your protein of choice purified. 4. How do you get your protein off the beads? Run a new buffer with extremely charged (ions like NaCl) substances to displace the proteins which will then elute out purified.

Secondary structure: How to determine

Circular Dichroism (CD): Samples of proteins absorb right and left circularly polarized light to DIFFERENT EXTENTS - To appear on a CD graph, feature must have CHIRALITY and a REPEAT PATTERN different CD spectra result from this analysis producing a graph where you can see alpha, beta, and random coil features.

Structure of alpha-keratin in hair

Coil-coil with one other alpha helix protein subunit! 2-chain very tight rope like structure, firmness of hair. 1. 2-chain coiled coil 2. Protofilament: lined up side by side and one after the other two-chain coiled coil

Chromatography purifies the protein... how do you checked it WORKED: Electrophoresis + SDS-Page how are proteins visualized? --- what can each tell you? - Coomassie blue stain - Western blot

Coomassie blue: Is my protein pure? Used to bind ALL proteins in a mix because it targets Arginine (R), H, K, and aromatic aa which nealry all proteins have. - less sensitive, requires at least 100 nanograms of protein from left to right you purify more and more and can see where only one band exists. Western blot: Is my protein present and how much? Can only bind a specific protein that has an antibody raised against it - blind to all other proteins in mix - VERY SENSITIVE, need much less protein QUESTION: in image it shows coomassie, what if it were a western blot? **** You would see one band all the way through showing the one protein you are looking into

Intrxn #2: Ionic Interaction How is it possible for water to dissolve ions if their bond enthalpy is HUGE?

Coulomb's law! εr = dielectric constant H2O has a super high dielectric constant which lowers the force of interaction between ions. Ions will then dissolve. - Water 'sheilds' electrostatic interactions/reduces by forming a barrier around/between the charges - Water reduces attraction between opposite charges - Water reduces repulsion between like charges

Chaperons! 2 functions Quiz question

Definition: Chaperones are proteins in which assist in taking other proteins to their native folded state NOT part of the final structure, they are the "enzymes/catalysts" of the folding process! TWO functions: 1. Chaperones prevent misfolding - Hydrophobic exposed regions to surface quickly aggregate with other hydrophobic material in cell, the chaperone will bind a FOLDING intermediate so the phobic region is not exposed to philic 2. Chaperones facilitate folding-- an ACTIVE form - A chaperon (like a barrel structure) will allow a protein to enter and use ATP-hydrolysis to facilitate proper folding to native state, has caps to lock in the protein. - GroES barrel! QUIZ ?: true of chaperones? FALSE: Chaperone class that acts as an enzyme to catalyze the protein folding chemical reaction FALSE: Chaperone class that decreases the free energy change of protein folding Correct! Chaperone class that actively facilitates folding Correct! Chaperone class that prevents misfolding and aggregation

H-bond continued-- Structure of ICE where do waters properties come from (hint = melting)

Each water molecules connects to 4 other water molecules via H-bonds and are directional! The strongest H-bond structure possible. This crytalline structure resulting is due to H-bond stability and makes it less dense than liquid water which is essentially sheets of H-bonded molecules which can move and slide past others. - water is free to move but there still re H-bonds! UPON MELTING: 15% of H bonds are broken but several remain - explaining liquid (and not gas) - explaining the high BP (must break ALL H-bonds) - explaining high heat capacity (hard to break H-bonds in the liquid water)

Analysis of protein sequencing: discovering primary sequences of a new protein 1. Edman Degradation (classical method) 2. Mass spectrometry (modern method) ... also can help determine? A. MW B. sequence

Edman: Ch3 slide 68 - successive rounds of N-terminal modification, cleavage, and then identification of the AA in the chain and continue all the way down - used to identify protein with KNOWN sequence METHOD:... add phenylisothiocynate to react with N-terminus, it will pull it off and you analyze the R-group... keep repeating this all the way down the chain Mass Spectrometry: method of choide - Can precisely identify the mass of a peptide, and thus the amino acid sequence, - can be used for POST-TRANSLATIONAL MODIFICATIONS too - AMAZING for determining the molecular weight, most precise up to the dalton decimal method 1--MW: Ionize your protein and then pass it through a electrical field, the movement through the feild will tell you its MW. Method 2-- aa seq: first the peptide is broken into fragmnents, ionized and fed into a spectrometer to find the starting molecular weight. Then, the fragment is fed into a Collision cell which uses high energy to break of the amino acids one by one! Your run the aa sequence back in the spectrometer and see the new MW, you can use a table of known aa weight values to see what aa was present that got kicked off!

Chromatography purifies the protein... how do you checked it WORKED: Electrophoresis + SDS-Page 1. HOW we separate proteins next card is how are proteins then visualized? 1. HOW we separate proteins next card is how are proteins then visualized?

Electrophoresis: electric field's move a protein from NEGATIVE to POSITIVE - moves proteins based on charge, size, and shapte - We only want it moved by SIZE... so... - Use SDS to denature and unfold proteins so they are ALL linear, no secondary or tertiary structure. Now experiment doesn't discriminate by size. - Also: SDS is an ion so it gives all proteins Uniformly negative charges so the repelling the negative electrode is constant among all the proteins... ONLY variable left is size! SDS: sodium dodecyl sulfate -- ionic detergent (micelles) .... will denature all proteins and make them negatively charged Page: polyacrylamide gel electrophoresis

Is solubility of salts enthalpy driven or entropy driven?

Enthalpy it cannot be, replacing a super strong bond with a H-bond, much weaker, so it must be ENTROPically favored by making more disorder lattice structure -> disorder in water ***NOTE: the water itself actually becomes ordered as it interacts with ions, but the ions leaving the lattice shape trumps that.

Discussion NMR vs XRay crystalography

NMR: shift of ATOMS, dissolved in solution X-ray crystallography: relates to electrons and x-ray diffraction!

Affinity chromatography -- the MOST common (90% labs)

Example from book: ATP-column chromatography 1. The beads in the column are polymers containing fixed ATP 2. Your protein of interest will then be run through the column and interact and bind to the ATP 3. How do you purify: Add a buffer filled with FREE ATP in high concentration and the protein will then leave/exit by interacting with buffer free ATP and not the bead. Real life example: Ni2+ and poly-histidine --- Ni2+ columns binds to poly-histidine tags. 1. Make protein of choice through bacterial insertion, however, you modify it to have a polyhistidine tag which allows it to bind Ni2+ 2. When passing your protein through Ni solution it will bind and not elute, purifying the protein buffer source. 3. How do you elute it? You cannot run Ni concentrated through or it unstabilizes your protein causing damage SO... you flow high concentration of IMIZIDOLE through and the protein elutes! Themes of elution: 1. use charges to pass through 2. Increase the [ ] of whatever it binds to as free and not on beads 3. Use whats known about what is in the structure. OTHER methods: -amylose columns (maltose binding protein MBP) - Glutathione column ( bind glutathione S-transferase (GST) tag)

Electrostatic interaction and coulomb's law

F proportional to q (charge) ... larger charges have stronger forces F inversely proportional to distance of charges Er = dielectric constant of solvent! TWO types of electrostatic interaction: 1. Hydrogen bonds: partial charges on atoms 2. ionic interaction: full charges on interactions

Two MAJOR classes of proteins based on TERTIARY STRUCTURE

Fibrous Proteins: Only used for physical texture. - Keratin: Hair, nails - Collagen Globular proteins: Most we are concerned with in life! - sphere, hydrophobic core and philic heads

Protonated/deprotonated acid and its conjugate base jargon for PROTEINS! pH > pKa ... relate proton. vs deprot. and vice versa

For proteins: Protonated = acidic deprotonated = conjugate base at midway point, pH=pKa ... and [protonated] = [deprotonated] pH>pKa ... [deprotonated] > [Protonated] pH<pKa ... [protonated] > [deprotonated] remember!!!! starting at pH=pKa is midway so equal amounts... if pH increases, more basic so more deprotonated, if pH decreases more acidic so more protonated.

Thermodynamics review:

Gibbs free energy: dG= dH - TdS the study of heat and energy exchange between a system and its surroundings. H = Enthalpy (heat) - make a bond = negative = exothermic T (kelvin) S = disorder or randomness - make a bond = increase order/lower disorder = smaller entropy - G > L > S - More molecules = more randomness = more entropy

Heme and its properties

Heme is a Prosthetic: non-amino acid compound that is often, not always, covalently bonded to some biological compound and needed for proper functioning! Heme has 6 coordination sites: 4 with the nitrogen of pyrrole rings, and 2 perpeindicular to ring system - for the HEMOGLOBIN: 5th coordination site is occupied with Proximal His The LAST coordination site, 6: Binds to oxygen distal, this distal oxygen then binds to DISTAL HIS Note***** The oxygen binds the heme with a kink and this allows the distal histidine to attach! Helps make CO less dangerous How? -Kink in O2 allows Distal His to attach and stabilize making bond more favorable (kink = favor 100 fold) - No kink in CO means no distal histidine will bind making it go from 10^5 times better binding heme down to 250x better. - NET effect: CO is still 250x stronger affinity to bind heme than O2 is.

Intrxn #3 Hydrophobic interactions:

Hydrophobic - fears water - non-polar, insoluble in water, soluble in non-polar solvents - DOES NOT form bonds with itself, instead it groups together to avoid touching water! examples include: - O2, CO2, long alipathic chains (fatty acids), phenyl groups, oils, lipids, wax, etc.

pH, Acids, bases, and buffers - equilibrium constant

Keq = equilibrium constant of ANY reaction! A + B <-> C + D ... Keq= [C] [D]/[A] [B] K is ONLY affected by temperature and related to the standard free energy change (dG) dG= -RT(Ln Keq) ; Keq = e^-dG/RT More negative dG -> larger K (more favorable = more products)

Micelles: forming them and why they do form example on how soap works How it ties into the lipid bilayer

In amphipathic molecules, there is a hydrophobic head and hydrophilic tail. The tails sequester from water and form a perfect spear with the hydrophilic heads on the outside which is known as a MICELLES! It does this to limit the exposure of non-polar to polar and limit a decrease in entropy. Soap and detergents are an example! - Oil and grease on our hands are hydrophobic and pass through the hydrophilic heads to the hydrophobic region... then when you wash your hands the grease leaves trapped inside the Micelles bubble with the water. - The Micelles can dissolve in water since it has hydrophilic head exterior. Lipid bilayer has tails inward since tails are too thick to form a sphere properly, so instead the edges fold over to form a large cylinder with a hollow inside!

Question iclick

Ka ... lower Ka then the lower affinity (higher Ka here should be protein A since need less [protein] to saturate Kd ... lower Kd then the higher affinity

Lecture 2-- Chapter 2 cont H-bond continued... Importance of Hydrogen Bonds in biomoleculues

Key role in acid/base of aqueous solutions... Essential for the structure and function of polypeptides, DNA, mRNA-tRNA (H-bonds needed for transcription), polysaccharides, and water (LIFE)!!!

Kw and pOH,pH calculations

Kw = [OH-][H+] = 1x10^-14 14 = pOH + pH pH = -log[H+] 7 = neutral, < 7 is acidic, > 7 is basic

Oxygen binding Hemoglobin and Myoglobin evolved to match physiological requirements

Look at graph, PO2 in tissues is 4, here myoglobin is nearly fully saturated. PO2 in lungs is 13, here Hemoglobin is nearly fully saturated so can pik up O2 and drop it off (about 38% of it) in the lungs at pO2 4. - 100% bound at pO2 13 -> 62% bound pO2 4 = 38% release Lungs: high affinity O2 for Hb, load up Tissues: Low affinity O2 for Hb, drop off on Myoglobin!

How can oxygen bind to Mb and Hb?

Metal ions (Fe2+ for example) are good oxygen biners, but free metal ions are reactive and produce oxidative stress... In Hb and Mb: Heme-- protoporphyrin ring with a central Fe2+ ... the 4 amino groups decreases reactivity of iron by allowing resonance of lone pair

Separation by Size (Size-exclusion chromotography)

Method and explanation: The matrix is made up of POROUS polymers so that the LARGEST protein has no way of entering the pores, so they simply just pass by the beads without entering the beads pores! The biggest protein elutes out first, the smaller proteins will get stuck in the beads porous and slow down due to friction, you need WAY more buffer (Velution) to get the protein to finally elute. Variables: Ve = SPECIFIC elution volume, amount of buffer needed to pass to get a protein to elute Vo = Void volume (exclusion volumne), constant based on given column Ve/Vo = relative elution volume... can be plotted against log MW to find the molar mass of that protein! KEY: The larger the molecular weight, the SMALLER the Ve - this is because larger proteins bass by beads without entering pores and getting stuck/experiencing friction!

Motifs vs domains

Motif: Small part of a domain structure, "super-secondary element" ... greater than 1 secondary structure (beta-alpha loop, etc.) Domain: Has a function on its own and forms its own 3D structure within a protein ... Tertiary structure! not quaternary!

Quaternary structure will 60 kDa polypeptide forming a homo-tertamer of 240 kDa migrate like 60 or 240? - on size exclusion - on SDS-Page

Multiple protein subunits aka PROTOMERS come together to form an interacting multisubunit protein. - Non-covalent interactions mostly plus disulfide covalent bonds called multimeric/oligomeric will 60 kDa polypeptide forming a homo-tertamer of 240 kDa migrate like 60 or 240? - SDS-Page: 60 kDa since the protein is UNRAVELED and the subunits are not really covalently bonded/stuck to the others DENATURATION loses 4˚,3˚,2˚ structure. - Size exclusion: move as a 240 kDa

Examples of REVERSIBLE ligand binding: structures overall Myoglobin (storage) Mb Hemoglobin (transport) Hb

Myoglobin: forms 3˚ structure, primarily found in MUSCLES, is a SINGLE polypeptide - 153 amino acids, 17 kDa structure: globin fold, heme is binding site for Oxygen, stores in muscle Hemoglobin: forms 4˚ structure, presen in erythrocytes (Red blood cells) - is a HETERO-TETRAMER (4 different subunits)

How to determine 3D structure: 1. X-Ray crystallography method, shortfalls, pros 2. Bimolecular nuclear magnetic resonance (NMR)

NMR utilizes the nucelar spin of a proton 1. Purify protien, dissolve the protein 2. Collect NMR data for ISOTOPES H1, C13, and N15 nuclear spins - regular naturally occuring atoms do not get picked up, so we have a way of expressing proteins with these isotopes 3. The protein then will show chemical shifts/NMR signals and using known shifts of certain bonds/atoms you can determine the structure. Pros: - NMR gives a MIXTURE of sturctures! It is a dynamic structure, tells which regions move and which are more static (comes out fuzzy) - No need to crystallize - Can see many hydrogens - Captures dynamics of protein structure Cons: - works best with SMALLLLL proteins... limited!

Determine if it is enthalpy or entropy driven? overall method

Need dG to be negative so its spontaneous. dG = dH -TdS .... ways to make a -dG -dH = exothermic, making stronger bonds than breaking or +dS = increasing disorder Which has the larger role, most cases they go opposite ALWAYS work independent of each other, if enthalpy is favorable, entropy will be unfavorable! ex: oxygen forming bond (O2) is favorable, enthalpy wins, cause it is entropically unfavorable (less disorder)

Organic Molecules and Functional Groups

Organic: molecules with covalently bonded carbon backbones - linear, branched, or cyclic carbon backbones Functional group: groups of atoms linked to carbon backbones (determine physical and chemical properties)

PHYSIOLOGICAL buffers in the body!

Our body likes to reside around pH = 7.4 --> 1. Phosphate buffer: in the cytoplasm and extracellular space H2PO4^- <-> H+ + HPO4^2- pKa = 6.86 - a good buffer since its in the buffer range! 2. CO2/bicarbonate buffer in BLOOD plasma bicarbonate (pKa 3.6, a badd buffer for the body) by itself is very unstable so you have to look at the entire system equilibrium instead! NET equation: CO2 (dissolved) + H2O <-> H+ + HCO3- - multiply Ka of each to get overall Ka... convert to pKa which is 6.1 ... a better buffer but not great so the body has mechanisms - CO2 DISSOLVED is the acid here - blood plasma pH has a bad buffer so is susceptibe to change! Slide 55: alternative ways for the CO2/bicarbonate buffer in blood is regulated by the body! A. adjust pH by respiration... CO2 exhaled from lungs B. Adjust by metabolism... H2CO3 <-> HCO3- + H+ - HCO3- leaves through digestive tract NH3 + H+ <-> NH4+ (removed in kidneys) NOTE*** multiply K values when combining two equations!

Dissociation constant Kd

P + L <-> PL P= Protein, L= Ligand ka= RATE association = [PL] / [P]*[L] kd= RATE disassociation = [P]*[L] / [PL] = 1/ ka **Higher the binding affinity, higher the ka, lower the Kd NOTE: ka = M^-1, kd= M Equilibrium of a assoc/dissaoc. Ka= [PL]/[P] [L] = ka/kd here Ka is eq. constant and ka is rate.

CHAPTER 4 start Levels of Protein Structure primary-- peptide bond basics and properties

Primary structure: Sequence of amino acids - structure of the Peptide bond: Alpha carbon, connected to H, alpha amino, and alpha carboxyl, and side chain - even though its tetrahedral, its PLANAR and rigid due to the resonance through the amino/carboxyl groups. It creates a partial double bond character. There then is a favored dipole in the TRANS configuration

How do proteins fold so fast? levinthals paradox Process (folding units)

Proteins fold in micro-milli seconds! Impossible for protein folding to occur by samling every possible conformation until lowest energy is reached = Levithal's paradox INSTEAD: direction toward the native structure is directed by thermodynamics towards most favorable native state. PROCESS: 1. proteins in red (short segments) form 'units' and fold independent of other units ... may have propensity to form certain secondary structure and so forth 2. Different folding units from 1. then interact with each other to form MOTIFS and DOMAINS and eventually the final tertiary structure. - since all folding units have propensity to form some structure, they all independently fold quickly and then quickly form 3d structure.

Hydrogen Bonds

Require three atoms in a line (DIRECTIONAL, linear, co-linear) ... 1. O/N/F 2. H and 3. O/N/f where 1/3 are donors and acceptors surrounding the H ("flanking" electronegative atoms) EN ATOMS: Cl, F, N, O, S, P, etc! Atom covalently bonded to H is the donor! Water can be both acceptor and donor! WHAT IF atoms are not colinear/directional? - if they are NOT colinear, the negative charged en^- atoms flanking the hydrogen will have repulsion making is thermodynamically unfavorable! This is why adenine/thymine have 2 H bonds and G/C have three!

Ligand Binding

Reversible, transient process of chemical equilibrium AB <-> A + B Molecule which binds is called a ligand (usually small, atom, molecule, functional group (could be a protein too)) to the ligand binding site via NON-COVALENT forces, hence transient

H-bond continued-- Solubility definition How does H-bonding help SOLUBILIZE polar biomolecules? (glucose) Is it enthalpy or entropy driven?

Solubility: ability of a solvent to interact MORE STRONGLY with the solute particles than the solute particles with themselves! - H-bonds help dissolve polar, uncharged compounds or even charged! How does H-bonding help SOLUBILIZE polar biomolecules? - Solute/solute H-bonds are replaced with water/solute favorable H-bonds within the glucose molecule's OH groups Is it enthalpy or entropy driven? - Breaking bonds is +, making stronger bonds is - ... we are breaking OH glucose/glucose hydrogen bonds but reforming glucose/water hydrogen bonds, basically the same thing! - Dissolving a solid/solute glucose causes a great increase in disorder and possible micro states. answer = ENTROPY

What happens when you place a hydrophobic compound in water? what about in another non-polar sovlent?

Solubilization of compound in water is thermodynamically unfavorable. 1. The Water (polar) does not want to touch the non-polar since it cannot form H-bonds! 2. The water forms a very ORDERED structure known as "CAGES" around the non-polar molecule to protect itself 3. This decreases entropy (less favorable) in order to counteract the lower energy, non-polar groups together so the surface area is minimized and there is less ordered water structures formed. ------So when you add one hydrophobic compound to a hydrophobic solvent, it immediately diffuses throughout the solvent as this will increase disorder and is hence entropically favorable.

Specific Activity to monitor PROTEIN PURITY

Specific activity = Protein activity of interest/total protein The higher the specific activity the greater the purity of the sample activity in "units" , total protein in mg.

To study proteins you really need them purified to their basic units...

Steps to protein purfication: 1. Grow cells that have modified to have added the protein of interest in their DNA to be transcribed/translated! (E coli) 2. Induce protein expression 3. Lyse the cells so all proteins are released into a protein buffer 4. Use CHROMOTOGRAPHIC Steps to purify protein of interest from other host cell proteins. THREE ways to separate/purify protein: 1. size. 2. Charge. 3. affinity for ligand 5. SDS-PAGE or enzyme specific activity monitors protein opurity.

pH Calculations of weak acids or bases HENDERSON HASSELBACH EQUATION what is a BUFFER

Still use Ka... but now it is not fully dissociated so you have to set up an equation ... [B][C]/[D] the pKa helps to determine the pH using titration curves! at the midpoint pH=pKa Henderson Hasselbach: used for a weak acid or base and its conjugate base or acid ... *BUFFER determinant +/- 1 pH*!!!! pH = pKa + log [A-]/[HA] Buffer: an aqueous solution that tends to resist changes in pH when small amount of acid or base are added! note**: SA/SB will not be a good buffer, either will be super high pH or low depending on what species is present

structural basis for Hemoglobins cooperative binding is explained by... - R and T state

T-State: tense, unable to bind Oxygen, low O2 levels "deoxy-Hb", low affinity conformation of Hb R-State: relaxed, able to bind oxygen and each binding increases affinity for oxygen, high O2 levels "Oxy-Hb"

Hemoglobin in adult vs fetus!

The fetus has to take oxygen from the blood of the mother, which makes for a kind of competition between adult and fetal hemoglobin. To make sure the fetus wins, fetal hemoglobin has a different structure (gamma instead of beta subunits) that can bind oxygen more strongly, making it able to "steal" oxygen from the mother's adult hemoglobin.

Protein folding tunnels - why afinsen got lucky

The folding funnel: - The most stable energy state is what is the NATIVE STRUCTURE - As seen, there are valleys where proteins might get stuck in and no be able to overcome the activation barrier.... in this case CHAPERONES help overcome barrier and properly reach native state. explains how Afinsen got lucky with RNAse!

Which structure is less unfavorable?

The grouped non-polar molecules since surface area is less, less cage so less order meaning you limit the drop in entropy... maximize the negative dG

Why is the size-exclusion chromatography not accurate in determining molar mass? rod like effective radius

The molar mass is only an estimate, calculating Ve/Vo on your graph with knowns and then adding your point on the X axis (Ve/Vo) can then give you the best fit line where it would correlate to the Y axis (MW). The reason its not accurate: Size eclusion depends on SHAPE... 1. Globular proteins 2. Rod like protein Globular proteins are pretty accurate with 'effective radius' ROD like proteins, rotate it in 3D and its EFFECTIVE radius becomes much larger than it really should be in one direction versus the other.

Tertiary structure: generally reveals FUNCTION and how it performs!

The overall spatial arrangement of atoms in a protein in 3D - structure formed when all secondary structures come together and interact - Stabilized by numerous weak interactions between AA side chains - Interacting amino acids do NOT need to be next to each other in primary structure to interact in tertiary!

Primary sequence continued, the rigid peptide plane!

The planar molecule CANNOT rotate around the peptide bond (amino/carboxyl) The molecule CAN rotate around the alpha carbon! - Phi: The amino group rotating, angle around alpha carbon - Psi: the carbonyl group rotating, angle around alpha carbon In a rigid, fully extended polypeptide, both phi and Psi are 180 degrees

Ribonuclease refolding experiment by Afinsen's experiment Conclusions

Unfolding: denaturation, dump in urea and lose protein activity Refolding: renaturation, remove urea and the RNAse function/3D structure can be recovered Conclusions: 1. Protein folding is spontaneous (dG < 0) - no energy needed to refold! Had to add something to unfold! 2. Protein folding is autonomous -- the folding 3D structure of the residues is written in the primary structure! -- got a little lucky, see later there are valleys proteins can get stuck in and not properly refold back to proper... = require chaperone!

SECONDARY STRUCTURE: 1. Alpha-helix -- Helical wheel representation and HELIX DIPOLE What stabilizes N-term?

Wheel representation: The inner diameter of a alpha helix is 4-5 Â, too small to fit anything - Residues N and N +7th are on top of each other - Residues N and N+ 3 are often forming H-bonds or Ionic interactions - The FACE of the wheel can have different properties! (hydrophobic, phillic, etc.) Helix Dipole: The amide has a partial H POSITIVE The Carbonyl has a partial O NEGATIVE - all peptide bonds have similar orientations make a LARGE macroscopic dipole moment! As a result of the dipole: Aspartate often stabilizes the Positive N-terminus

How to determine 3D structure: 1. X-Ray crystallography method, shortfalls, pros 2. Bimolecular nuclear magnetic resonance (NMR)

X,Y,Z coordinates for every atom = 3D structure 1. Purify your protein 2. 100s of Crystallization methods to crystallize your proteinterm-75 3. Shoot with X-rays to collect diffraction data map which will be analyzed by computer! 4. Upon computers making the map, you will get meshes of electron density maps, since you know the primary structure of the protein and bond angles of protein, you can fit your protein into the map and solve the crystal structure. Pros: - NO SIZE LIMITS - Well-established, automated maps produced SHORTFALL: - POLAR Hydrogens have basically no electrons so cannot be registered! - Requires crystals which are REALLY hard to get

Separation by charge: relation between pH, pI, and charge DETERMINING acidic vs basic proteins

acidic amino acids: pI < 7, so is neagatively charge at pH = 7 Basic amino acids: pI >7, so is positively charged at pH=7 determining acidic/basic proteins: 1. acidic: D and E >> R and K ... NEGATIVE charge at pH =7 since acidic, less than 7, so deprotonated 2. Bacis: R and K >> D and E ... positive charge at pH=7 NOT histidine 3. Charge of the protein depends not only on types and number of acidic and basic side chains, but also on the pH stated ******Higher number gets the proton******

Acids/bases and CA/CB Ka of strong acids and bases.

acids: proton donors (HCl, H2SO4) Bases: Proton acceptors (NaOH) Ka is the dissociation constant of an ACID: HA<-> H+ and A- ... specific type of Keq STRONGER ACID = LOWER pKa = HIGHER Ka = LOWER pH ***Note: Strong acids/bases fully dissociate

Pathway for bicarbonate in the body

after formation of unstable bicarbonate it forms dissolved CO2 and H2O .... CO2 can be respirated out through lungs

LECTURE 3: ch 2 cont ICKLICK on patient toxin (hypo) ... metabolic kidney compensate

answer: B alkalosis 1. acidosis -> 2. Neutral -> 3. *secondary alkalosis* KEY: Respiratory changes are very FAST, metabolic changes are very SLOW

Iclicker

answer: B is false. Hydrophobics NEVER form bonds - ENTROPY driven, not enthalpy driven D is true, cage forms and lowers entropy

Metabolic and respiratory acidosis metabolic acidosis: cause, response, possible treatment Respiratory acidosis: cause and response

blood pH turns less than 7.35! Metabolic acidosis: - severe diarrea, lose HCO3- (CB) so body turns acidic - RESPONSE: Rapid and deep breathing (rid of CO2 to make more basic) - treatment: mass doses/pills of bicarbonate given Respiratory acidosis: - CAUSE: impaired pulmonary function- HypOventilation: dissolved CO2 builds up in the blood - Response: secrete NH4+ acid through kidneys, body turns more basic (get rid of lots of H+)

Proteins are found as...

coenzymes, cofactors, prothetic groups, other modifications

Protein stability and folding! dG? why is this good? folded vs unfolded enthalpy or entropy? Function depends on .... denaturation definition How to denature

dGfolding: -20 kJ to -65 kJ... about the same strength/favorability as a single hydrogen bond (SLIGHLY favorable) = able to denature/renature FROM PERSPECTIVE OF THE PROTEIN: Unfolded state: High entropy, weak interactions between amino acid R groups and surrounding water - all conformations available are possible for phi/psi, greater entropy Folded State: Many weak non-covalent interactions (H-bonds, ionic interactions, van der waals) and covalent disulfide bonds - locked Phi/psi combos = lower entropy Enthalpy vs entropy driven: - ENTHALPY driven: when folding, you make order so negative dS means enthalpy bond making must be driving this process. The protein function depends on the 3d structure - Loss of structure (means also loss of function) = DENATURATION Caused by: - Heat or cold - pH change - Organic solvents - chaotropic agents: Urea and Guanidinium hydrochloride (GdnCL/GdnHCL) (chaotropic agent weakens H-bonds)

Stereoisomers vs isomers vs diasteromers vs enantiomers vs conformational vs configurational

isomers: molecules that share the same chemical formula! Stereoisomers: molecules with the same formula but different SPATIAL conformation configurational isomers: Must break a bond in order to superimpose the molecule! Is a DIFFERENT molecule. TWO TYPES of configurational isomers-- 1. cis-trans stereoisomers (geometric isomers): require C=C bonds, same side more substituted = cis example: Retinal in eye with light converts cis->trans allows us to see! 2. enantiomers and diastereomers: chiral carbon substituted with 4 different items - enantiomers = NON-superimposable mirror images (RSSR - SRRS) ... plane polarized (5 degrees CW, enan is 5 degrees CCW). - NON mirror images = diastereomers Conformational isomers: identical molecules just rotate around a bond - most stable = staggered (less steric hindrance) - least = eclipsed

How to calculate pI of an ionizable side chain amino acid

left pka to zwittern + right pKa to zitter / 2 NOTICE: K and R pI >> 7 since basic moves towards 14 D and E pI << 7, acidic (- charge) move to 0

Lysine and Arginine titration curves

pK1 is from from pK2, but pK2 and pKr are close to eachother pKr comes LAST since they are so basic of side chains!

Histidine titration curves

pK1, pK2, and pK2 are equidistant basically from each other.

Formation of amino acids into peptides/proteins titrations of proteins with multiple ionizeable groups

peptides: <100 aa proteins: >100 aa Reaction: Condensation... - amino group of one aa reacts with carboxylic acid of another aa forming a dimeric unit KEY: You take two ionzieable groups (carboxylic acid and amino) and destroy them, no ionizeable groups left! - ALSO: forms an amide! cannot be prot./deprot -- whats left is still a pK1 and pK2 When combinging, titrations work the same, order not important but what R groups are there! Make sure to look for K, R, H, E, and D

Column Chromatography

protein buffer passed through matrix, separate and exit as the ELUATE (what is collected at bottom)

CHAPTER 3!!! Proteins and Amino acids

proteins: main agents of catalysis (enzymes like DNA polymerase, enolase), transport nutrients (hemoglobin, lactose permease), structure (collagen and keratin, motion at cellular level (myosin/actin). Amino acids: building blocks of proteins, heteropolymers (20 types of aa make up polypeptide/protein) that may or may not have covalernt modifications. well suited for: - polymerization, acid-base properties, pysichal properties, chemical functionality ****Note: put amino and H group on wedge out of page, if the amino group is LEFT it is L (not D) -*all nturally occurring aminos are L config*

Why Myoglobin is good for storage of O2? is it good for transport?

rectangular hyperbola graph! Myoglobin has a very high Ka (low Kd) so needs small pressures of O2 (not concentration since O2 is a gas) to reach saturation! - Kd = .4 In the lung pO2 = 13 kPa In the tissues pO2 = 4 kPa Regardless, its about 90% bound O2 regardless of location... so it is a GREAT STORAGE molecule of oxygen since it binds even in low pressures. Why isn't it a good transport: - Bad because it won't ever really release O2 at homeostatic or life condition levels

FUNCTIONAL GROUPS L1 slides 24-26

review them in the exam 1 cheat sheet notebook on ipad!

The limited conformational space between peptide bonds in proteins results in SECONDARY STRUCTURE: 1. Alpha-helix 2. Beta-sheets Perfect helix? held together by? (how many apart and which part of the aa) 2 turns = ... right or left handed Peptide bonds parallel or perp... r group?

secondary sequence: Pattern in space based on a given amino acid sequence Alpha Helix: helical backbone is held together by hydrogen bonds between the Nth carbonyl oxygen and the N+4 amide nitrogen - there are 3.6 residues (5.4 Â) per turn of the helix - 2 turns about 7 amino acids - Tetrahedral carbons make it so the angles do not allow perfect helix, but pretty good. Note: always runs N-> C The alpha helix is RIGHT handed ("L") in all natural occurring Amino acids - each peptide bond is PARALLEL to the helical axis - Each R group os perpendicular to the helical axis

Binding curve determining Kd and its meaning from a curve - lower =?

θ = [L] / ([L + Kd) **when [L] = Kd, you are at θ=.5 meaning 50% ligand are bound to protein RECTANGULAR HYPERBOLA shape Kd = the ligand concentration at which 50% of the binding sites are occpied - The lower the Kd, the less ligand is needed to reach 50% saturation and therefore, the stronger affinity binding. Lower Kd = less [L] required to saturate protein = higher affinity Higher Kd = more [L] required to saturate protein = lower affinity