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Hardin BIOCHEM Test 1

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Overview of Biochemistry
Overview of Biochemistry
Energy
sources include:

Adenosine Triphosphate (ATP)

Reduced Nicotinamide Adenine Dinucleotide (NADH)

Reduced Coenzyme Q (Ubiquinone)

Phosphoenolpyruvate
Endoplasmic Reticulum (ER)
Rough ER - rough due to the presence of ribosomes (ribosomes synthesize proteins)

Smooth ER - does not contain ribosomes; produces cellular products like hormones/lipids
Ribosomes
synthesize proteins --> also a variety of post-translational modifications occur here (i.e disulfide bond formation, addition of carbohydrate/lipids, acetylation)
Golgi apparatus
A system of membranes involved in post-translational modifications and packaging proteins for export/transport by the cell

Transport vesicles transport modified proteins
Mitochondria
produces adenosine triphosphate (ATP) --> through a process called oxidative phosphorylation and driven by a PROTON gradient which is generated as a result of the electron transport process

Evolutionary Origin:
--Derive from blue-green bacterial cells with their own chromosomes that were captured by other cells and subjugated

--Mitochondrial DNA inherited from one's mother
Mitochondria Continued
Reducing equivalent carriers such as nicotinamide adenine dinucleotide (NADH) and reduced coenzyme Q (CoQH2) provide the electrons/reducing power to drive electron transport
Chloroplast
light driven ATP production which is coupled to electron transport

Carbon fixation occurs in the Calvin Cycle Pathway

Key features include: grana, thylakoid membrane, stroma

Evolutionary origin: derive from the same blue-green bacterial cells (same as mitochondria)
Other Organelles
Cytoskeleton - protein network consisting of actin filaments, intermediates filaments, microtubules; dynamic

Membranes - form separate compartments that limit and direct the trafficking of biomolecules and crucial for signal transduction/active transport/etc.
pKa's
Depend on environments of functional groups

--Measures affinity (bonding strength) of functional group binding to a proton

Small pKa = LOW AFFINITY

High pKa = HIGH AFFINITY
Water
2 Electron lone pairs bond with other waters (bond with another's hydrogen)

Electronegativity produces overall molecular dipole
Polarity
polarity of water produces hydrophilicity in which polar solutes are soluble in the solvent

Oxygen, nitrogen, and metals are polarizable which leads to ionic/polar bonding with water

pH --> H+ concentration that ranges to 14 orders
Hydrogen Bonding
Hydrogen bonds with N, O, H etc.
Amphipathicity
composed of both hydrophilic and hydrophobic parts
ATP (adenosine triphosphate)
Why ATP is energy carrier?

Because it has HIGH GROUP TRANSFER POTENTIAL!
Oxidation/Reduction
A(red) + B(ox) --> A(ox) + B(red)

Electron transfers from A(red) to B(ox)

Not just loss of hydrogen, but also a loss of an electron
Van der Waals interactions
Weak attractions between molecules or parts of molecules that result from transient local partial charges.
Peptide Bond formation due to....
dehydration
Osmotic Pressure
pressure that must be applied to prevent osmotic movement across a selectively permeable membrane

PV = nRT --> P= (n/V)RT = cRT --> ∆P = ∆cRT

--Molecules and H20 can pass through semipereable membrane (higher concentration will go to low concentration to balance out both sides)

--Only H20 can pass through the semipermeable membrane, water will move to more concentrated side

Molecular weight cutoff is 12K Dalton
Isoelectric point (pI)
pH at which molecue has charge of zero

Look above at protonated molecule, and then see how many charges are there! say if 2, then go to 2 on axis and draw over until it hits curve, this is the pI

or

Average the two pKa groups to get pI
Factors that Influence pKa of protonatable/deprotonatable groups
1) Dehydration

2) Charge-charge interactions - positive charge --> decrease in pKa

3) Charge-dipole Interactions - hydrogen bonding --> increase pKa
Proteins
Polymers composed of a-amino acids linked by peptide bonds

N terminus --> C terminus

Nomenclature: "-ine" replaced by "-yl"
exceptions:
Asparaginyl, glutaminyl, cysteinyl, tryptophanyl
Purification and Characterization of Proteins (next 5)
Purification and Characterization of Proteins
Purification of Proteins
Solid-phase chemical synthesis - binding reactants to ligand which is bound to an insoluble bead held inside a column

Typical Approach to Purification:
1) Grow cells so protein largely present

2) Disrupt cell membrane

3) Remove insoluble debris by centriguging cell homogenate

4) Separate debris from crude sample

5) Preserve catalytic function by including additives

6) Add ammonium sulfate to salt out desired protein
Gel Filtration Chromatography
smaller molecules are retained longer while larger molecules elute earlier through the gel
Ion Exchange Chromatography
column matrix modified with cation/anion ligands (negative charge would bind to cation ligand)

More negative molecules requires higher salt to be dislodged
Ligand-dependent Affinity Chromatography
use ligand/enzyme binding abilities to purify
Electrophoresis
current carries charged proteins, voltage provides energy for movement, resistance provided by gel as proteins migrate from upper negatively charge to lower positively charged reservoir

Smaller = faster

Larger = slower through gel
Amino Acid Sequence Determination (next 2 slides)
Amino Acid Determination
Edman Degradation (AA Sequence Determination) [see photo in phone!!]
Uses PITC to label every AA from protein

Involves making pITC derivatives of AA

Step 1: Hydrolysis of protein to get individual AA's

Step 2: Reaction with N-terminus of protein with PITC

High pressure liquid chromatography identifies/quantifies the Amino Acid
Sanger's Reagent
Uses 1-fluoro-2,4-dinitrobenzene to react with N-terminus of AA and used for sequencing
X-Ray Diffraction Analysis
light diffracted by electron density of an array of proteins embedded within a crystal --> this produces a specific pattern of spots on an imaging apparatus and can then calculate structure of protein
Protein Structure
Protein Structure
Classification of Substructure
1st level of classification involves degree of compatibility with water

1) Water soluble - many enzymes, typically globular in shape

2) Water insoluble - structural materials (keratin for hair/fingernails)
Primary Structure
linear sequence of covalently linked amino acids
Secondary Structure
Regular 3-D folded structures found within proteins

Includes:
a-helix
antiparallel/parallel B-sheets
U-turns
Tertiary Structure
Complete folded structure of single subunit

Packing and stabilization of interconnected substructures from a single chain into the overall globular structure
Quaternary Structure
Packing of subunits into multisubunit complex

i.e. hemoglobin which has tetrameric quaternary structure
Domains
portion of a protein that does a specific function

within the Tertiary Structure

ex. Rossman fold - where ATP binds in a protein
Alpha Helices (α-helix) [Secondary Structure]
-Right handed due to steric interference between carbonyl oxygen and amino acid side chains

-Helix conformation reinforced by hydrogen bonding between each carbonyl oxygen and the amide hydrogen of 4th amino acid toward C-terminus

-For one complete turn of α-helix, 3.6 AA residues required (13 atoms away)
Beta Sheets [Secondary Structure]
Form when two or more polypeptide chain segments either fold back upon themselves (anti-parallel) or line up side by side in the same direction (parallel)

Sheets stabilized by hydrogen bonds between amide hydrogen and carbonyl groups of adjacent chains

ex. Rossman fold or beta barrels
Parallel Beta Sheets
Polypeptide chains arranged side by side in same N- to C- terminal direction

Hydrogen bonds evenly spaced but SLANTED
Antiparallel Beta Sheets
MORE STABLE!

Run in opposite direction with respect to N- to C- terminal

Hydrogen bonds PERPENDICULAR to the strands and space alternates from wide to narrow
Pleated sheets
when parallel/antiparallel are mixed
U-turns [Secondary Structure]
Proline exchanges between cis and trans structures

Cis form supports formation of u-turns resulting in reversal of the direction of the protein chain
Ramachandran Plot
shows coordinates corresponding to a variety of protein conformation

Each (psi, phi) coordinate pair gives coordinate for one amino acid

Plot consists of all data pairs for a given protein

Used to characterize the details of the structure of a protein after its structure has been determined (i.e. X-Ray Crystallography)

Each dot corresponds to a specific AA
Stabilizing Factors of Proteins
1) Hydrophobic Effect

2) Hydrogen Bonding

3) Disulfide bonds

4) Van der Waals Forces

5) Dipole-dipole interactions

6) Ionic Bonds
Thermodynamics of Protein Folding: Hydrophobic Effect
Thermodynamics of Protein Folding: Hydrophobic Effect
Hydrophobic effect
Balance between two energies: intrinsic entropy of the chain (always favors unfolding) and enthalpy accrued by hydrogen bonding of water molecules surrounding the macromolecule

this is the: PRIMARY DRIVING FORCE OF PROTEIN FOLDING
Gibbs Free Energy for Protein Unfolding
∆Gu = +∆Hu - T∆Su

where ∆Su = entropy (affected by # of configurations)

∆Hu = enthalply (gained by hydrogen bond formation in water molecules surrounding protein)
example
if ∆Gu = +1 --> unfolding is non spontaneous/ folding is spontaneous

∆Gu = -1 --> unfolding is spontaneous,

∆Gf = +10 --> folding is nonspontaneous (unfolding)
Temperature Dependent Denaturation
Unfolding more spontaneous due to increase in temperature which affects contribution due to entropy

Temperature multiplies by entropy which can outweigh the enthalpy and lead to unfolding
At X Temp
1) At low Temp (20°C) the protein is stable

2) At higher T (>70°C) protein denatures/unfolds
Why?
Protein folds/unfold depending on what happens to the surrounding water (enthalpy)
At low temperatures
Hydrogen bonds among surrounding water molecules are intact

Protein always wants to unfold because it becomes more disordered (entropy) but hydrogen bonds overcome that tendency


and thus encaged protein folds to small volume to maximize H-bonding
At high temps
Hydrogen bonds drive apart by more vibrational energy

T is multiplied by ∆Su and the tendency to unfold is enhanced

--also the many released water molecules gain entropic energy releasing the folded prtein from its cage allowing it to unfold

--> THUS enthalpy is overcome!
Factors that Affect Proteins
Chaotropes (enthalpic term)

Disulfide Bonds (manipulate entropic term)

Temperature (entropic term)

Kosmotropes (enthalpic term)
Hofmeister Series
propensities of salts to induce denaturation or foster folding
Chaotropes
cause chaos/disrupt binding; disturb the hydration cage around the protein

Favor protein denaturation/unfold to the entropically favored denatured form

Examples includes urea, sodium dodecyl sulfate, salts like NaClO4 (sodium perchlorate) and guanidinium
Sodium Dodecyl Sulfate (SDS)
chaotrope

Amphipathic compound that has polar head and nonpolar chain

Disrupts hydrophobic effect and SDS then hydrogen bonds with solution leading to eventual protein unfolding
Kosmotropes
Foster protein folding

Ammonium cation and sulfate dianion (NH4)2SO4
Undergoing Folding Back to Original Condition
Molten globule - partially folded protein intermediate state found during denaturation; often have some intact secondary structural features but a yet to be stabilized interior structure

--with small proteins folding is cooperative and no intermediates

--with large proteins a molten globule is formed first followed by return to native form

--Even larger proteins with multiple domains fold separately in each domain, ending with whole molecule returning to native form
Molecular Chaperones (Chaperonins)
Assist with folding/assembly of proteins

Most are classified as heat shock proteins (hsp) --> stabilizes unfolded proteins

Can renature by removing urea and oxidizing sulfhydryl groups back to disulfides
Size Exclusion Chromatography (GEL Filtration)
Includes sepharose beads that contain KOSMOTROPES (sulfate group) so protein won't unfold (proteins maintain native form)

Bead contains tortuous network of fibers

--Smallest beads will get in and out of beads and kept from going out of column (smallest come out last)

--Large beads don't go in bead at all and goes straight through the column (come out first)
size exclusion chromatography
SMALLEST BEADS COME OUT LAST

LARGEST BEADS COME OUT FIRST!!!!
Gel Electrophoresis
Have to have 1) Buffer 2) SDS 3) Gel

Sodium Dodecyl Sulfate (SDS) - unfolds protein

About 2 sodium dodecyl sulfates per amino acid

1) Protein becomes linearized since protein is unfolded thus leading to the negative charge of protein

2) Top reservoir is negatively charged, bottom reservoir is positively charged
--use a dye (coomassie blue) for staining to identify protein

--> SMALLEST PROTEINS COME OUT FIRST THROUGH THE TORTUOUS NETWORK`
Ligand Binding and Functional Control
Ligand Binding and Functional Control
heme
porphyrin ring composed of four pyrrole rings (Fe 2+ held in the center)

Used by myoglobin/hemoglobin (specifically the iron) to bind oxygen
Globular proteins
contain cavities/clefts whose structure is complementary to the structure of a specific ligand; clefts usually contain ionizable residues that aid in binding

holoprotein - cofactor + protein

apoprotein - cofactor absent
Myoglobin
binds O2 in muscle tissues

ONLY BINDS ONE O2

Composed of eight alpha helices

Heme group alternately binds iron and oxygen reversibly

Hyperbolic curve on graph
Hemoglobin
binds moelcular oxygen (O2) and transports it from lungs to tissus within red blood cells

Has a tetrameric quaternary structure with FOUR O2 binding pockets containing one heme group each

AGAIN: contains four subunits where each contains a heme group that binds reversibly with oxygen

--central cavity contains six positively charged side chains

Sigmoidal curve on the graph
Hemoglobin binds
O2 with lower affinity than myoglobin (P50 is higher than that of myoglobin)

Hemoglobin nearly saturated with oxygen in lungs where pO2 is high
Cooperativity in O2 Binding
Hemoglobin displays sigmoidal curve which indicates cooperativity

While the hyperbolic curve of myoglobin does NOT
Positive cooperativity
With hemoglobin, binding the first O2 makes subsequent molecules bind more easily -->

when O2 binds first site, next binding site changes making it more susceptible to ligand binding


Hemoglobin is positive cooperativity in site 1 to 2 but negative cooperativity in site 3 to 4 because at low O2 it picks up O2 efficiently but at higher O2 it tries to release O2 (mixed cooperativity)
2,3-bisphosphoglycerate (BPG)

(Glycolysis Intermediate Derivative)
Inhibits Binding of O2

BPG is an allosteric effector because it binds to central cavity in tetrameric complex at a site other than the oxygen binding site
BPG
1) When hemoglobin is in deoxy conformation, positively charged groups bind to negatively charges of BPG (stabilizes deoxygenated form of hemoglobin)

2) When hemoglobin is oxygenated, Beta chains closer together, thus allosteric binding site is too small to bind BPG

3) In absence of BPG, hemoglobin has higher affinity for oxygen

4) Hemoglobin transfers oxygen to myoglobin at low partial pressures of oxygen in tissues

5) At high pO2 of oxygen in lung alveoli, and absence of BPG, hemoglobin binds oxygen

BPG concentration is an indicator of the status of the metabolic pathway - where there's more ATP, glycolytic compound 3-phosphoglycerate is converted to BPG which limits amount of O2 bound by hemoglobin
Bohr Effect
the effect of H+ on the binding of O2 to hemoglobin; H+ is an allosteric effector which like BPG favors deoxygenated hemoglobin
What do Allosteric Effectors Change
the inhibitor/activator modifies binding protein which changes the Ka of the binding equilibrium
Buffering of Blood (Bicarbonate System)
Dissolved CO2 in the blood + H20 leads to H2CO3 (carbonic acid) using a carbonic anhydrase catalyst

Then it oxidizes (loses H+) to become HCO3 (bicarbonate) which then oxidizes again to become CO3(2-) [carbonate]

At pH 7, bicarbonate dominates
--for the buffer, pH = -log[H+]free
pH = -log[H+]free
Having the free H+ available for molecules to take up does not change pH, which is essentially a buffer

A buffer to keep blood pH at the physiological range

Thus if you add enough bicarbonate then it pushes system in typical physiological pH range
Van der Waals
+ sign = how close the atoms are and the repulsive force

- sign = how far atoms are and the attractiveness

once it reaches 20 Angstrom, there is no attraction

smaller Dielectric constant means higher interaction between atoms
Edman Degradation Further Explained
Method of sequencing amino acids in a peptide

Technique 1: Hydrolysis by heating the protein in 6M hydrochloric acid to 100-110 degrees celsius for24+ hours which will subsequently provide constituent amino acids

Technique 2: Includes a reaction with the N-terminus of the protein; phenylisothiocyanate (PITC) is reacted with the N-terminus amino group andthen cleaved and treated with an acid which is then identified by chromataography/electrophoresis
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