Biochem Ch 8 & 9

• Scientific reasons- to study protein function
• Therapeutic purposes- i.e production of insulin
• Use of enzymes in pharmaceutics and industry

• Identify an organism/organ/tissue that produces a protein of interest in "large" quantities
• Homogenize the tissue and extract your protein together with thousands of others
• Perform numerous selective separation steps until the protein is isolated to sufficient purity

• Identify a gene of interest, isolate DNA, amplify by PCR, clone to a plasmid
• Over-express the protein in an appropriate organism: bacterial cells, yeast, insect
• Break open the cells to extract protein and perform selective separation steps until the protein is isolated to sufficient purity

• Solubility 
• Ionic charge
• Polarity/hydrophobicity
• Size
• Binding specificity

• Molecules with a net charge will migrate in an electric field with a velocity proportional to:
• Charge density, size and shape

Generally used as an analytical technique, occasionally as a preparative technique

Constrain ions to move within a solid support (such as a thin gel between glass plates) to minimize diffusion

• Top and bottom buffer reservoirs are connected by a porous gel
• Top reservoir is typically connected to the cathode; the bottom to the anode

The electric field; the two chambers are only connected with the gel so protein must go through

• Acrylamide and methylenebisacrylamide
• Used in PAGE

• Concentration of acrylamide
• Degree of crosslinking (concentration of methylenebisacrylamide)

• The mixture of macromolecules will go in porous gel
• Once you run the electrophoresis then the large proteins tangle in the gel and have slower migration

• High: better separation of small proteins
• Low: large proteins
• Gradient of density: both large and small

• Denaturing gel electrophoresis
• SDS: detergent with C12 hydrocarbon chain (hydrophobic) and a negatively charged sulfate (highly polar)
• SDS bonds to hydrophobic regions of protein side chains

• SDS denatures protein tertiary structure by disrupting internal charge and hydrophobic interactions -> uniform shape
• Overwhelms protein native charge -> uniform negative charge
• All proteins now have a uniform charge/mas ration

Mobility of proteins towards the positive electrode (anode) depends only on their size

• SDS: denaturing detergent; unfolds and coats proteins with negative charge
• Reducing agents (beta-mercaptoethanol; dithiothreitol, TCEP): break any disulfide bonds and prevent random formation of bonds between different proteins
• Blue dye: to visualize the solvent front
• Glycerol: increases density of sample, so it drops to the bottom of the sample well buffer to maintain pH
• Boil for 3-5 minutes to ensure maximal unfolding

Reduction of covalent protein disulfide bonds

• Using standard proteins with known molecular weights (MW) to calibrate
• Then measure and plot distance migrated of protein standards vs. log of their MW
• Draw a standard curve through the linear range and measure the distance migrated of your unknown
• Locate the corresponding log MW and find the MW of the unknown protein

Complexes with the unfolded proteins (detects ~50 ng)

Improves sensitivity by ~50 times to ~1 ng

• Detect a specific protein by staining with antibody 
• Sensitivity depends on antibody, but can be way below 1 ng

• SDS-PAGE typical example
• Denature proteins with SDS and heating, break up complexes, break down disulfide bonds, then run gel
• Use to show what proteins are present in a mixture

• Run electrophoresis without SDS/head denaturation to maintain folded proteins/complexes/associations
• Due to difference in charge, proteins might run in opposite directions or not at all
• Often is used to demonstrate interactions (protein-protein, protein-nucleic acid interactions)
• Can be reducing or non-reducing

• The pH at which the protein carries no net charge
• Technically, pI= 1/2(pK₁+pK₂) for transitions around neutral

• When pH > pI protein is negatively charged
• When pH < pI protein will be positively charged

The minimal solubility of a folded protein come from this because of interactions with water as a solvent

• Will be equal to the average between the pKa values of ionizable groups
• 

When it is beyond ~1 pH unit

• If a protein contains 4 Arg, 10 Glu, 3 Asp, 2 Lys and no other charged AAs
• it will have 6 positive charges, 13 negative charges

• Electrophoresis in a pH gradient
• Molecules run in a pH gradient until they reach the pH equal to their pI

• Experimental determination of pI of a peptide or protein
• To separate proteins/peptides based on the difference in their pI values

• At a very low pH, all molecules are positively charged
• As (+)ve ions move towards the (-)ve pole, the pH increases and they become less protonated until at their pI, the net charge is 0
• As (-)ve ions move towards the (+)ve pole, the pH decreases and they become more protonated until at their pI

• Used to: 
• Separate complex protein mixtures
• Detect posttranslational modifications (PTM) i.e phosphorylation can strongly affect protein pI and therefore can be easily detected
• 1st dimension-IEF
• 2nd dimension-PAGE

Powerful methods for protein purification and characterization based on differential interaction of proteins (in mobile phase) with a solid column matrix (stationary phase)

Uses interactions between a protein mixture (mobile phase) and a solid support (stationary phase) for separation based on a single physical property including size, charge, hydrophobicity, specific interaction

The process of flowing the protein out of the column

• Stationary phase (column, matrix, beads):
• Ion exchange
• Gel filtration
• Reverse-phase
• Affinity

Mobile phase (solvent, conditions required to elute protein) depends of protein and column used

• Electrostatic interactions between a charged protein (above or below the pI) and a charged stationary phase
• A positively charged protein (basic) will bind stronger to a negatively charged matrix (acidic matrix)
• A negatively charged protein will bind stronger to a positively charged matrix (basic matrix)

• Allows for the separation of proteins based on size and shape (also known as size exclusion chromatography)
• Solid phase: beads with pores of a defined shape and size
• larger proteins pass through a smaller volume (only between the beads) where smaller proteins can get into beads which makes for separation and a longer path
• Different resins target different weight

Hydrophobic stationary phase (hydrophobicity-based separation)

• Unrelated to target protein
• Sizes range from a few AA's to entire fusion proteins
• Fusion to N-terminal or C-terminal end of target protein

• 6-10 His in a row bind tightly to nickel beads
• Eluted by addition of imidazole (looks like His side chain)

• Maltose binding protein binds to amylose (linear chain of glucose) resin
• Eluted by addition of maltose (glucose-based disaccharide)

• The enzyme glutathione-S-Transferase (GST) binds tightly to glutathione (a tripeptide)
• Eluted by addition of excess of free glutathione

• Separation base on specific interactions (i.e enzyme-substrate; protein-ligand; protein-metal)
• Unwanted proteins are washed through the column and protein of interest is eluted by ligand solution
• Kids have "higher affinity" to playgrounds than adults do, and can be separated from adults by passing them through playgrounds. Then, kids can be eluted from the solid matrix by passing other kids who have not played yet.

Positively charged (basic) matrix that interacts with anions

Negatively charged (acidic) matrix that interacts with cations

• Choose pH where protein has an appropriate charge
• Proteins will bind with beads through electrostatics
• Salt (Na or CL) will compete for interactions and exchange the ions
• Use salt gradient to promote release (elution) of the bound proteins
• 

• A concentration gradient of salt causes fractionation, and separation of multiple protein species based on strength of interaction (red>>purple>>yellow at a particular pH)
• High concentration of salt will elute all proteins from the column
• 

• To separate proteins with at least 2x size ration
• Can separate 15kDa from 30kDa
• Probably not 30kDa from 45kDa

• Another size-based approach
• Removing small molecules (salts, buffer, etc) by diffusion via semi-permeable membrane with small pores while retaining proteins inside the dialysis bag

Provides information about content, not the sequence

• How many chains (quaternary structure)?
• Break protein down into short peptides
• Analyze peptide sequences
• Combine all of these into a primary sequence
• Special consideration: disulfide bonds

• Proteins are too long to be sequenced at once
• Peptide bonds can be specifically cleaved by peptidases/proteases

Breaks peptide bond

Breaks peptide bond somewhere in the middle of (inside) the sequence

Breaks peptide bond on a terminal amino acid ("exterior" peptide bond)

Exopeptidase that hydrolyzes peptide bond at the C-terminus (carboxyl terminus)

Exopeptidase that hydrolyzes peptide bond of the N-terminal residue (amino terminus)

• Carboxymethylation
• Cys irreversible modification is required to prevent oxidation

• Reducing disulfide bonds
• Using DTT Cleland's Reagent
• 

• Basic residues
• Cleavage points: lys, arg

• Aromatic residues
• Cleavage points: phe, trp, tyr

Cleavage points: asp, glu

Cleavage points: ASP, GLu

Lys (C)

• Chemical cleavage
• Cleavage points: met (C)

• Make it possible to determine the original sequence of the polypeptide chain
• Combine overlapping sequences together

• 
• Phenylisothiocyanate (PITC) is the reagentq

• Ion source, Mass analyzer, detector
• All separation is based on mass to charge ratio

• Protein is supplied in a volatile buffer (i.e ammonium acetate)
• Evaporation of a drop in vacuum results in charge repulsion and dispersion to smaller drops
• Generates population of protein ions with many charge states

• Mass reconstruction/deconvolution
• 

• Peptides are mixed with matrix:
• absorbs laser energy
• often acids (source of H+)
• small size to not interfere with peptides
• Not volatile in vacuum
• Relatively polar to dissolve in water solutions

• Ionized biomolecules are separated in electric field
• Mass can be determined from time it takes to reach the detector: time of flight (mass is directly proportional to time
• Fewer charge states

• Electrospray MS: syringe full of protein/peptide solution
• Ionize by concentrating charges in vacuum
• Highly charged spectrum
• Often connected to liquid chromatography systems
• Can be used to analyze "native" proteins

• MALDI MS: crystallize protein/peptide in matrix
• Ionize w laser
• Simpler analysis of mixtures of ions
• Samples (proteins/peptides) are mixed with matrix and dried
• Typically denatured samples

• Defining protein identity by masses of its peptides
• Many different peptides have nearly identical masses but the combination of masses is unique for each protein
• 

• Digest protein into peptides
• MS1 observes different digested peptides- this alone can determine identity of the protein (peptide finger printing)
• Select desired peptide (instrument control) break amide bonds per molecule in collision chamber
• MS2: observe the series of peptide fragments
• Use the known residue masses to determine sequence