BCH210

Biochemical molecules of life (lec 2)

* Proteins (antibodies, receptors, transporters, hormones, enzymes)
* Lipids (fats, cholesterol, hormones)
* Sugars (nrg, cell struct)
* Nucleic Acids (DNA, RNA)
* Small molecules (vitamins, metabolites)
* Ions (K+, Na+, Ca2+, Cl-)
* Water (H2O)

Structure is key in Biochemistry (lec 2)

* biochem is the study of chemical processes within living organisms
* structure of protein/sugar/lipid is important for it's function
* cellular diseases arise when a genetic mutation affects the structure and function of a protein
* proteins play a variety of roles including catalysis, structure, and recognition

Proteins are the action molecules of the cell (lec 2)

* enzymes, transporters, lipoproteins, hormones, signaling molecules, receptors, recognition molecules, glycoproteins, structural proteins, motility proteins

How does binding and hydrolysis occur (lec 2)

* binding and hydrolysis occurs once substrate and enzyme recognize each other
* can be recognized through structure or cofactors

Proteins contain diff types of bonds (lec 2)

* covalent bonds hold together amino acids
* non cov allow chains to fold into final struct
* cofactors may be bound cov/noncov
* transporters may also bind molecules non-cov
* chem func groups are responsible for mediating this binding

Protein cofactors (lec 2)

* non proteins molecules (organic/inorganic) that assist w/ struct and/or func
* 2 types: (can also be loosely or tightly bound to protein)


1. essential ions (inorganic)
2. coenzymes (organic)

Diff types of interactions (lec 2)

Hydrogen bonding (lec 2)

* strong attractive non-cov forces
* acceptors: EN atoms (has lone pairs)
* donors: H atoms covalently bound to EN elements (O, N, F, Cl, Br)

Functional groups (lec 2)

* can be ionized, nucleophiles/electron acceptors, H-bond acceptors/donors
* __**amides:**__ important for protein struct
* __**Phosphate:**__ important for regulation (very polar)
* __**Phenyl:**__ important for R group (amino acid side chains), impedes ability to form H-bonds (resonance)

Water - very important molecule (lec 2)

* can form up to 4 __transient__ H-bonds due to unequal sharing of electrons (dipole)
* important for molecule solubilization and formation of complex struct

Molecule solubility (lec 2)

* “like dissolves like”
* water can solubilize molecules that have hydrophilic func groups (polar)
* amphiphiles: both hydrophobic/philic

Hydrophobic effect (lec 2)

* non polar molecules aggregate in aq solution (form together exclude water)
* main driving force behind formation of macromolecular struct (helps w/ protein folding
* seems to contradict 2nd law of thermodynamics (entropy (disorderness) always increases in spontaneous rxn)
* but decrease of entropy of struct causes increase in water’s entropy (leading to overall increase in entropy

Water and biochem molecules (lec 2)

* water is an excellent nucleophile and can participate in hydrolysis and condensation rxns
* depending on how macromolecules fold, rxns could be prevented from occurring but maximizing # of interactions of hydrophilic func groups w/ H2O for solubility

Biochem and drug design (lec 2)

* drugs come in many diff forms and delivered in diff ways
* binding interactions + intermolecular forces are important considerations
* presence/absence of func groups can assist w/ solubility (water/lipids) and binding (H-bonds, ionic, hydrophobic)) to specific targets

Macromolecules (tut 1)

* large molecules from linked, repeating units (monomers to polymers)

Major categories:


1. proteins
2. carbohydrates (most complex, variety of subunits + branched struct)
3. nucleic acids

Diffs. btwn macromolecules (tut 1)

1. basic units: (a.a., sugars, nucleotides)
2. connection bond: (peptide, glycosidic, phosphodiester)
3. structure: linear vs. branched

Amino acid struct (lec 3)

Made of:

* amino group (NH3) (+)
* carboxy group (COO) (-)
* R side chain

*peptide bond made in condensation rxn btwn amino group and carboxyl group*

Chiral amino acids (lec 3)

* asymmetric alpha carbon centre results in chirality
* chiral molecules: non-superimposable, mirror images (enantiomers)
* isomers exhibit optical activity
* __**L amino acids are physiologically relevant**__ in plant and animal proteins

Zwitterions (lec 3)

* a neutral molecule that has separate positively and negatively charged functional groups
* R side chain needs to be uncharged for amino acid to be zwitterons

Amino acid metabolism (lec 3)

* can be metabolized to form other important molecules including:
* hormones
* neurotransmitters
* nitrogenous bases (DNA/RNA)
* Energy-producing intermediates

Amino acids and protein structure (lec 4)

* 20 amino acids have distinct side chains that contribute to proteins struct and func
* non-cov/cov interactions btwn func groups r improtant for holding protein together + its ability to interact w/ other molecules
* interaction of hydrophillic amino acids w/ H2O help solubilize some proteins

Non-covalent interactions (lec 4)

* __**H-bonds**__ can form btwn hydroxyl, carboxyl, thiol, and amino groups to __**help w/ protein solubility**__ (*if on surface of protein*)
* H-bonds can also form btwn amino acid side chains w/in protein’s struct (every 4 residues) (folds into more compact struct)
* hydrophobic interactions can occur btwn aliphatic + hydrophobic side chains
* __**ionic interactions**__ are important for __**ligand, cofactor and/or substrate binding in enzymes**__
* salt bridges can form btwn positively + negatively charged amino acids

Disulfide bonds (lec 4)

* covalently cross-link two cysteines together
* disulfide bridges can be intrachain/btwn diff polypeptide chains
* these linkages can __**stabilize struct**__
* Protein Disulfide Isomerase (PDI) enzymes help catalyze this oxidation rxn

Secreted vs. cytosolic proteins (lec 4)

* disulfide bond formation is post-translational modification that occurs in some secreted proteins as they pass through ER
* cytosolic proteins usually contain cysteines due to reducing nature of cytosol
* disulfide bonds can be broken by reducing agents in cytosol or lab

Post-translational modifications (lec 4)

* addition/removal of func groups can change struct of protein and affect its func and/or degradation
* disulfide bond formation is type of post-translational modification
* important covalent modifications:
* phosphorylation
* ubiquitination (proteins tagged w/ ubiquitin for degradation)
* glycosylation
* acetly, methyl, hydroxyl, carboxyl
* cofactor/ligand binding also important for struct/func

GFP and amino acid modifications (lec 4)

* cyclization of tripeptide (Ser65-Tyr66-Gly67) followed by dehydration and oxidation leads to formation of green fluorophore
* amino acid changes can influence absorbance spectra

Amino acid properties (lec 4)

* amino acid sequence determines 3D struct of proteins and can reveal important info abt proteins evolutionary history
* can be used to elucidate proteins func using homology searches
* mutations in primary sequence can lead to change in func or disease
* are conserved for struct and/or func

pH (lec 5)

* H+ and OH- ions are formed upon dissociation of H2O/other weak acids (H+donors) or bases (H+ acceptors)
* pH = -log \[H+\]
* at lower pH values, more H+ are present to protonate diff func groups
* pH of solution important since func groups can also act as weak acids/bases, losing + accepting H+ at diff pHs

pKa values (lec 5)

* used to measure strength of acid (HA)
* proton removal of weak acid HA is described by:
* Ka = (\[H+\]\[A-\])/\[HA\], pKa = -log Ka
* strong acids have high Ka (high dissociation) and low pKa
* pKa value tells you the pH at which func group loses/gains its H+

Henderson-Hasselbach eqn (lec 5)

* used to determin pH of solution depending on the amount of ionization of weak acid (protonated \[HA\] vs. deprotonated \[A-\])
* derived from acid dissociation constant eqn (Ka)
* pH = pKa + log (\[A-\]/\[HA\])
* can be used to determine pH of mixture of weak acid and conjugate base

pH + pKa values (lec 5)

* pKa is pH at which weak acid/func group is 50% protonated and 50% deprotonated
* proteins contain func groups that must be protonated/ deprotonated to allow for non-cov interactions to take place
* buffers r solutions of weak acids + conjugate bases that can resist changes in pH, maintaining protein’s struct and/or func
* buffers can neutralize small additions/loss of H+, keeping pH of solution stable
* buffers maintain pH +/- 1 pH unit around pKa

Choosing the right buffer (lec 5)

* select buffer that has pKa closest to desired pH at given temp
* consider chem stability (are there any chem rxns that may occur producing molecules that will affect your protein, cell or experiment)
* does buffer interfere w/ any of your experiments
* cost/availability?

DIff cellular environments (lec 5)

* Physiological pH: 7.4
* Blood pH: 7.35-7.45
* Stomach: 1.5-3.5
* Intracellular pH:
* cytoplasm: 7.2-7.8
* lysosomes: < 5.5
* golgi: 6.0-7.9
* mitochondria: 7.8
* some cells may also survive in acidic/alkaline environments

Titration of histidine (lec 5)

* His has 3 func groups that can be protonated/deprotonated
* 3 buffering regions exist around 3 pKa values
* isoelectric point (pl) is pH when charge of molecule is zero

pH and buffers are essential in biochem (lec 5)

* proteins contain many func groups essential for formation of non-cov interactions, crucial for protein’s struct and/or func
* pH also affects H-bonding that is important for enzymatic func and ability to interact w/ H2O and other binding partners
* chem environment of func group can influence pKa value
* buffers are important for maintaining stable pH environment in order to study protein/biological process

Blood’s buffering ability (lec 5)

* normal (physiological) pH of human blood is 7.4
* bicarbonate is main buffering species, accepting and donating H+ to prevent changes in pH
* CO2 is exhaled to remove excess H+

Bohr effect (lec 5)

* pH can affect oxygen carrying ability of hemoglobin
* when CO2 is made in tissues and combines w/ H2O to make bicarbonate and H+, this helps O2 release
* at lower pH, His146 is protonated and creates a salt bridge to Asp94
* favours the deoxygenated struct of hemoglobin

Essential vs nonessential Amino acid (tut 2)

• Essential:

  • amino acids can’t be produced by body and need many chemical reactions to be made

  • must come from food sources

• Nonessential:

  • can be produced by body, easily made from intermediate metabolites

Protonation state (tut 2)

• pH < pKa = protonated

• pH = pKa = 50% mix of both species

• pH > pKa = deprotonated

Levels of protein structure (lec 6)

1. Primary (deg. 1): linear sequence of amino acids encoded by DNA

2. Secondary (deg. 2): periodic regular structures (alpha helix, beta strands/turns) (H-bond interactions form these structs)

3. Tertiary (deg. 3): folding of secondary structures into define protein motifs and domains

4. Quaternary (deg. 4): assembly of distinct chains into multi-subunits structures

Primary structure directionality (lec 6)

• amino acids are joined enzymatically in condensation rxn

• polypeptide chain has directionality, amino terminal is start of chain

• backbone consists of peptide bonds and alpha carbons, while variable part are distinct R side chains

Peptide bonds (lec 6)

• are planar

• polar but uncharged

• have partial double-bond character due to resonance, preventing rotation of peptide bond

Polypeptide chains (lec 6)

• peptide bonds are planar but rotation can occur around alpha carbon (amide to carbon, carbonyl to carbon)

• angle range from -180 deg to 180 deg, but not angles are permitted

• steric clashes are minimized when side chains are trans to one another

Secondary structures (lec 6)

• arise from non-cov interactions btwn func groups

• alpha helix

• beta strands and sheets

• beta turn

alpha helix (lec 6)

• right handed helix w/ side chains pointing out

• intra-strand hydrogen bonds form btwn backbone C=O and N-H groups (i and i+4), buried down centre of helix

• there 3.6 residues per 360 deg turn and each residue is 1.5 A high

Properties of alpha helix (lec 6)

• properties depend on side chains

• amphipathic most common

Beta strands and sheets (lec 6)

• more extended structure where intermolecular H-bond link 2/+ beta strands to form beta strands

• H-bonds occur btwn carbonyl and amines

• strands may be parallel, antiparallel(shown in picture) or mixed

• can bring distant parts of protein together

• dimensions are more extended, allowing for diff interactions to occur

• adjacent R groups alternately point up and down causing each side of the sheet to have diff properties

Beta pleated sheet (lec 6)

Beta turns (aka reversed turns) (lec 6)

• join 2 beta strands to form sheet

• 4 residue segment that allows peptide chain to turn 180 deg

• can be found on surface of globular proteins, connect secondary structures

• H-bonds form btwn carbonyl O and amine H

• Pro (P) is common at position 2

• Gly (G), Asn (N), Ser (S) also seen frequently in turns (N and S have good modifications which make them more common)

Higher levels of organization (lec 6)

• secondary structures come together to form stable, 3D tertiary structures called motifs/larger domains linked by flexible linker segments

• domains may function independently of the rest of the protein

• ligands may assist by bringing distinct regions together

• disulfide bonds can stabilize both tertiary and quaternary structures

• quaternary structure involves arrangement of multiple subunits of distinct polypeptide chains

• tertiary structure is final lvl if only 1 polypeptide chain

Frequently seen motifs (lec 6)

• coiled coil (2 helices wrapped around each other)

• helix bundle

• beta alpha beta unit

• hairpin

• Greek key

• beta barrel (sheet wrapped so pore forms)

• zinc finger motif

Quaternary structure (lec 6)

• multi-subunit proteins may consist of identical/non-identical polypeptides held together cov/non-cov

• diff subunits may arise from multiple genes/due to post-translational cleavage of precursors

• larger macromolecules can also form due to interactions btwn polypeptide chains

• very complex

How do proteins fold? (lec 6)

• secondary structures form due to favourable H-bonding

• non-cov interactions (H-bond, ionic, VdW) and disufide bonds play key role in tertiary and quaternary struct

• hydrophobic effect is primary driving force in protein folding (forming of more stable structures)

• random coils not necessarily random, they may be stable structures

• chaperones prevent aggregation of newly synthesized and unfolded proteins by binding to exposed hydrophobic regions (transient interactions)

Protein structure and drug design (lec 6)

• knowing a protein struct at all levels can help design molecules that interact and inhibit protein's func

• inhibitors can be designed to mimic an enzyme’s substrate but prevent rxn from occuring

• drugs can also block and prevent binding interactions btwn proteins necessary for signaling or a downstream event, or even modify amino acid side chains and change a protein’s struct

• molecules can also block necessary non-cov interactions required for structure (ex: chelators of metal binding)

Protein purification (lec 7)

• cell is a crowded environment w/ ~ 10^10 proteins per mammalian cell

• in order to study a protein’s struct and/or func, we need to be able to purify it from other cellular components

• other molecules/ions/proteins may interfere/modify your protein, creating a heterogenous population

• ability to purify protein is first step in understanding its struct and/or func

preparation of crude extract (lec 7)

• choosing correct cell/tissue source is important for obtaining adequate quantity and quality of sample

• purifying intracellular proteins require lysing cell to generate crude extract consisting of mixture of proteins and cellular contents

• centrifugation can be used to produce a supernatant of soluble materials and pellet other large organelles/insoluble precipitants

• molecular bio can also be used to genetically engineer your protein of interest

Cell lysis (lec 7)

• mechanical/physsical methods:

  • grinding

  • sonification

  • vortexing w/ glass beads

• osmotic pressure (water flows into cell and breaks it apart)

• chemical bases (ex: detergents)

Extra considerations:

• lysing cells may release proteases, enzymes that may cleave your protein of interest

• conditions (pH/temp) may alter your protein’s structure and lead to denaturation

Types of chromatography (lec 7)

• Chromatography: diff partitioning of molecule btwn mobile (buffer) and stationary (column) phase

• proteins can be purified based on diff in chemical properties

  • Size/shape: Size-exclusion/gel filtration chromatography

  • Charge: ion exchange chromatography

  • binding interactions: affinity chromatography

  • hydrophobicity: RP-HPLC (Reverse Phase High Pressure Liquid Chromatography)

Size exclusion chromatography (lec 7)

• proteins are separated on basis of size

• column containing a resin of porous beads allow smaller proteins to enter the beads, while larger proteins are excluded and exit first

• elongated proteins may appear larger as their tumble through buffer and also be excluded, eluting faster

• calibration w/ proteins known of MW is required

Protein absorbance (lec 7)

• biomolecules absorb light at characteristic wavelengths ad this can be measured using spectrophotometer

• most proteins are colourless and don’t absorb visible light (380-750 nm)

• concentration of protein in solution can be measured based on absorbance at 280 nm, arising from aromatic amino acids

• stains like Coomassie Blue can also be used to visualize proteins/quantify protein concentration in Bradford assay

Beer-Lambert Law (lec 7)

• E: molar extinction coefficient (1/Mcm) depends on # of Trp + Tyr (Phe-minimal)

• c: concentration (M)

• I: path length of cuvette (cm)

• A = log (Io/I)

Size-exclusion chromatography example (lec 7)

• Vo: void volume, anything larger than column’s fractional range goes straight through

• Ve: elution volume of molecule

• Vt: total volume of column

Ion-exchange chromatography (lec 7)

• separates amino acids/peptides based on net charge

• pl = pH when polypeptide is neutral

• cation exchange resins binds (+) charged peptides while anion exchange resins attracts and binds (-) charged polypeptides

• proteins can be eluted by increasing salt concentration/changing pH

Affinity Chromatography (lec 7)

• proteins bind column based on their affinity for specific molecules/chemical groups

• resin contains covalently bound molecules/ligands that recognizes certain proteins in mixture and interacts via non-cov interactions

• bound protein is released by passing solution containing free molecules to compete for binding

• useful for concentrating proteins

Example of affinity chromatography: His tags and nickel-NTA resin (lec 7)

• histidine side chains can bind to Ni2+ when bound to NTA (nitrilotriacetic acid) resin

• His tages: 6-10 His residues can be added to proteins to help w/ purification

• free imidazole can be used to elute protein of interest

Dialysis for protein purification (lec 7)

• can be used to remove small moleules

• high salt may interfere w/ other experiments/assays

• pH of buffer may also (de)protonate side chains involved in chem rxns/interactions

• buffer exchange can be done to change pH of buffer as well

High Pressure Liquid Chromatography (HPLC) (lec 7)

• uses very fine beads and high-pressure pumps to move sample through column achieving higher resolution of peaks

• resin choice determines separation basis, usually silica covered in HCs

• aka Reverse Phase PHLC when separating based on hydrophobicity

• in RP-HPLC, hydrophobic compounds interact stronger w/ column and have longer retention time

Determining purity of sample (lec 7)

SDS-PAGE (lec 7)

• Sodium Dodecyl Sulfate (SDS) - PolyAcrylamide Gel Electrophoresis (PAGE)

• SDS denatures proteins w/ 1 molecule of SDS binding every 2 amino acids (amphipathic)

• proteins will have same charge: mass ratio and migrate in gel towards anode

• polyacrylamide gel creates mesh/sieve of cross-linked molecules that separate subunits based on size

• size can be deduced by comparing MW markers

Amino Acid composition vs. sequence (lec 8)

• composition of protein could be obtained by hydrolyzing peptide bonds and quantifying amino acids

• however, may not tell much abt the protein unless it has unique composition (ex: gelatin)

• structure of protein may not necessarily tell what protein is present as many share common domains

• one way proteins can be identified is through amino acid sequencing

Protein sequencing (lec 8)

• sequence could be obtained indirectly via DNA sequencing, however, post-translational modifications may occur

• samples must first be purified before sequencing can occur

• Edman degradation can be used to sequence amino acids from N-terminus and carboxypeptidase for C-terminus

Edman degradation (lec 8)

• series of chem rxns

• N-terminal labeled w/ PLTC

• low pH to remove peptide bond

Limitations of Edman Degradation (lec 8)

• limited to ~100 amino acids

• post-translational modifications may block N-terminus and other complementary techs may be required

• proteins can contain thousands of amino acids and smaller fragments may need to be generated, purified and then sequenced

• Other chemicals and proteases can be used to generate smaller fragments for Edman degradation

Enzymatic and chemical cleavage (lec 8)

Protein cleavage example (lec 8)

• 15 amino acid polypeptide is treated w/ trypsin to generate the following 3 peptides, sequenced using Edman Degradation:

  • EH QSVVWK and AVFNDYR

• cleavage w/ chymotrypsin generates following 4 peptides:

  • KEH NDY RQSVVW and AVF

• what is sequence of og polypeptide:

  • AVFNDYRQSVVWKEH

Identifying peptides (lec 8)

• smaller fragments can be sequenced using Edman Degradation

• SDS-PAGE and immunoblotting (Western Blotting) can be used to identify larger peptides containing specific sequence using antigen-specific antibodies

• Polypeptides can be ionized, separated, sequenced and identified based on their mass: charge ratio using (Tandem) Mass Spectrometry

Immunoblotting (western blotting) (lec 8)

• proteins are separated by SDS-PAGE and transferred to solid-support membrane (blotting)

• primary antibody, specific for protein of interest, is added to recognize either linear sequences of amino acids

• secondary antibodies are specific for Fc domain of primary antibody and are attached to fluorescently labeled tag/enzyme that generates chemi-luminescent product

• will reveal whether or not protein of interest is present in sample and it’s potential size based on where it migrated on gel

Mass spectrometry (lec 8)

• peptides are bombarded by laser/high energy electron beam to create ionized fragments

• products are attracted to charged plate detector and analyzed in mass analyzer by their time of flight

• their time of flight depends on charge and mass of molecule

• comparison to known peptides can elucidate mass and sequence of polypeptide

Sequencing using Tandem Mass Spectrometry (MS/MS) (lec 8)

• used to determine sequence of amino acid

Sequencing using Mass spec (lec 8)

Studying Purified Proteins (lec 8)

• function:

  • enzymes assays

  • inhibition of function

• Interactions:

  • binding interactions

  • transport assays

• structure

• expression

Tryptophan Fluorescence (lec 9)

• presence of indole ring also allows Trp to fluoresce when excited w/ UV light (270-295 nm)

• emission of Trp occurs btwn 310-355 nm and is sensitive to polarity of its local environment in protein

• in a polar environment, fluorescence is ‘red-shifted’ to longer wavelengths and be less intense (opposite for ‘blue-shifted)

Protein Chromophores (lec 9)

• chromophore groups usually contain conjugated double bonds

• chemical groups absorb ultraviolet (UV) and/or visible light at characteristic wavelengths (can give proteins colour)

• aromatic rings and amide carbonyls are important chromophores found in proteins

• protein’s structure influences accessibility of these groups to light and can be used to characterize protein’s struct

Protein structure techniques (lec 9)

Localized primary and secondary structure:

• Fluorescence spectroscopy

• Infra-red (IR) spectroscopy

• Circular Dichroism (CD) spectroscopy

overall 3D structure:

• X-ray crystallography

• Nuclear magnetic resonance (NMR) spectroscopy

• Electron microscopy (EM)

Infrared (IR) spectroscopy (lec 9)

• proteins contain vibrating, strecthing, and bending groups

• these motions lead to an absorption of infrared radiation

• most prominent bonds, N-H and C=O groups in peptide bond, contribute most to absorption seen

• IR spectrum an be used to understand secondary structures that are present and influence H-bonds

H-bonding and IR Spectra (lec 9)

• band position of amide I band (C=O stretch, NH bend) can distinguish alpha helix and beta sheet structures

• stronger H-bonds, weaker C=O bond, lowering position of IR spec.

Circular Dichroism (CD) (lec 9)

• asymmetry of proteins results in diff (molar ellipticity) in absorption of left and right circularly polarized UV light

• chiral alpha carbons and secondary structures preferentially absorb 1 direction of light over the other

• protein folding and secondary structure content can be assessed based on observed spectra:

  • alpha helix: 222nm and 208nm (negative), 195nm (positive)

  • beta sheet: 217nm (negative)

  • Random coil: 198nm (negative)

• Studies proteins in solution to stimulate cell environment

CD experiment - Antibiotic peptide (lec 9)

• CD spectra was determined in presence of water (aq) or sds, which mimics the hydrophobic environment of membrane

• transition from random coil to alpha helix can be seen

• what interactions are mediating this change?

X-ray crystallography (lec 9)

• some proteins can form ordered crystals under varying conditions (pH, high salt, etc)

• crystals placed in X-ray diffractometer produce diffraction patterns that be interpreted in terms of atomic positions (x,y,z)

• the (𝜙, Ѱ) angles of each residue defines protein fold

• 3D structures of proteins can be reconstituted at high resolution (2A), including backbone and side chains

• structures are deposited in Protein Data Bank (PDB)

Electron density maps and resolution (lec 9)

Nuclear Magnetic Resonance (lec 9)

• carried out on proteins in solution

• used to be limited to smaller proteins (<25 kDa), but now can be done on larger proteins

• can monitor conformational changes, proteins folding and interactions w/ other molecules

• NMR is based on nuclear spin of certain nuclei (¹H, ¹³C, ¹⁵N) that can be measured in a strong, static magnetic field

• absorption of electromagnetic radiation can be used to deduce environment of nucleus and determine protein’s structure

1D NMR of ethanol vs larger protein (lec 9)

2 NMR Spectra of protein (lec 9)

Cryo-electron microscopy (lec 9)

• larger complexes can be visualized (>100 kDa)

• thin layer of protein solution is prepared on fine grid and frozen (cryo) very quickly to trap molecules in ensemble of orientations

• high powered microscopes measure beam of electrons that pass through protein sample. Diffraction in beam can be used to elucidate structure

• structure of single particle can be obtained and multiple structures are averaged out to build 3D representation of protein

Studying proteins experimentally (lec 10)

• understanding protein’s physiological environment is important for designing experiment that mimics cellular conditions (temp., pH, detergents, cofactors/substrates, etc.)

• experiments can be done in test tube (in vitro) w/ purified proteins, or in live cells cells (in vivo) to determine its intracellular localization/binding partners

• assays (experiment) can take advantage of non-cov interactions, chemical catalysis and other biochem properties to elucidate protein’s function

• control experiments are just as important when analyzing data

Important biomolecular interactions (lec 10)

• interaction of protein w/ protein/molecule is crucial for its function and numerous biochem processes:

  • protein folding/unfolding

  • cellular localization

  • post-translational modifications

  • signalling pathways and regulation

  • metabolism

• complication: non-cov interactions may be transient and short lived

Studying biomolecular interactions (lec 10)

• in addition to structural studies, common techs used in biochem:

  • Protein-Protein interactions: Co-immunoprecipitation and pull-down assays, cross-linking reagents, 2-hybrid screening, FRET

  • Protein-molecule interactions: transport assays, enzymatic reactions

• visual output is most common way of detecting occurence of biomolecular interaction

• enzymes and fluorescent proteins can also be exploited to generate secondary visual response when used as a reporter (ex: luciferase, beta-galactosidase-based assays, bioID)

Pull-down Assays (Co-immunoprecipitation) (lec 10)

• Question: does protein X interact w/ protein Y in cells?

• Approach: pull down X w/ and see if Y comes along

• Tools required: Antibody specific to X + antibody specific to Y

• Experimental steps:

  • immobilize antibody to X to a solid support (ex: beads)

  • break open cells and mix w/ antibody

  • isolate beads, wash and detect bound proteins by Western blot

Detecting binding partners by immunoblot (lec 10)

• often to be sure, you would do the experiment in reverse: pull down Y and probe for X on the blot

Capturing Complexes w/ chemical cross-linking (lec 10)

• chemical cross-linking can form cov bonds btwn molecules

• cross-linking studies can be used to reveal the inter and intra- (molecule organization of amino acids based on their locations)

• Formaldehyde is an example of non-specific chemical crosslinker

• cross-linking can also be nonselective using photo-reactive groups

• reactive side chains of amino acids can be targeted specifically for cov bond formation:

  • primary amines

  • carboxyls

  • carbonyls

  • sulfhydryls