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179 Cards in this Set

  • Front
  • Back
What is the "Progenote"?
A breakthrough organism which gave rise to everything; LUCA - last universal common ancestor
What were the prebiotic energy sources?
- Radiation from sun
- Electric discharge (lightning)
- Radioactivity
- Volcanoes
What were the initial raw materials on Earth?
CO2, CH4, N2, H2, H2O, NH3
What molecule is abundant in many structures which are necessary for life? Why?
Adenine - When concentrated HCN is refluxed, the major product is adenine; therefore abundant prebiotically
How do new enzymes/proteins begin?
Gene duplication yields an extra copy that can evolve with a new function
What is phosphotriesterase?
A molecule which is found in bacteria that hydrolyzes pesticides and nerve toxins; evidence of the quick evolution of enzymes
Evo-Devo stands for what?
Evolutionary development
What percent of the typical cell is water?
What leads to Hydrogen Bonding in water?
Electrical asymmetry; partial positive on hydrogens, partial negative on oxygen
How does hydrogen bonding affect melting points?
More hydrogen bonds, higher melting point
Which is the H-bond donor?
H-bond acceptor?
Donor = Hydrogen involved
Acceptor = Atom bonded to Hydrogen
The hydrophobic effect causes what change in entropy?
ΔS = Positive, because H bond potential is disrupted by hydrophobic molecules
What are Van der Waals interactions?
Weak interactions which operate at close range between transient dipoles induced in each atom by the other
The stronger the acid, the ______ the pKa; why?
Stronger acids = lower pKa = lower pH --- higher likelihood to ionize
What is the Henderson Hasselbalch Equation?
pH = pKa + log [A-]/[HA]
How does the Greek lettering system work for R groups?
αC = carbon attached to amino and carboxyl group
β, γ, δ, ε, etc carbons go consecutively downwards
What is the difference between configuration and conformation?
- Configurations - can be interconverted only by breaking and reforming one or more covalent bonds
- Conformation - Interconverted by rotation about a bond without breaking a covalent bond
What are stereoisomers which are nonsuperimposable?
Which enantiomer of amino acids is found in proteins?
L-amino acids
Which amino acids absorb UV light?
The isoelectric point is the pH when the amino acid is?
at a neutral charge
Which amino acids were found in the prebiotic atmosphere?
Glycine, Alanine, valine, leucine, isoleucine, proline, serine, threonine, aspartate, glutamate
Which amino acid kinks protein structure?
Which amino acid has a neutral pKa and is involved in H+ transfers?
Which nonstandard amino acid is involved in blood clotting?
Which nonstandard amino acid is a pre-cursor for arginine?
What is the average pKa of COOH on amino acids?
What is the average pKa of NH2 on amino acids?
How are peptide bonds formed in vitro (test tube)?
1) Attachment to resin
2) Removal of protecting group (Fmoc)
3) Activation of next amino acid
4) Peptide bond formation
5) Removal of polypeptide from resin
What is the purpose of Fmoc?
It is a protecting group which prevents unwanted reactions at the α-amino group of the residue
How does chemical synthesis (in vitro) of proteins differ from that done naturally (in vivo)?
In vitro - synthesis proceeds from the C-terminus to the N-terminus; reverse of the In vivo method (N-->C)
When naming peptides, start at which end?
If the molecular weight of a peptide is <10,000 it is designated as?
>10,000 is what?
<10,000 = Polypeptide
>10,000 = Protein
What is the average weight of an amino acid (adjusted for the percentage at which the amino acid occurs in proteins)
MW = 110
What are some functions of proteins?
- Catalysis
- Transport
- Nutrient / Storage
- Motion
- Structure
- Defense
- Regulation
- Many other miscellaneous
99.9% of reactions have what?
An enzyme catalyst
What are two examples of transport proteins?
Ex: O2 doesn't diffuse very quickly; hemoglobin provides transport
Ex: Lipoproteins
What are the main components for motion in muscle?
Action / Myosin
What is the main protein involved in structure?
What are examples of kinds of proteins involved in defense?
What kind of proteins are involved in regulation?
Hormones with extracellular signals
Why do some proteins have carbohydrates attached? Name?
Glycoproteins - used to identify cells
Why do some proteins contain metal cofactors? Name?
Metallo-enzymes - 1/3 of enzymes have metal cofactors
Why are lipids sometimes on proteins?
Helpful to embed in membrane
What are two ways to detect proteins?
1) UV Absorption (tryptophan especially)
2) Colorimetric Assays
- Lowry Blue Complex
- Bradford Blue Dye (Coomassie)
(Color intensity proportional to amount of protein)
Why is the Bradford Blue Dye (Coomassie) better than Lowry Blue Complex?
Coomassie doesn't destroy the proteins in the process?
What is the reaction rate proportional to?
The amount of enzyme present
What is the "international enzyme unit"?
The amount of enzyme that will convert one μmol of substrate to product in one minute at 30˚C
What are the units of activity?
Units (of desired protein) per mL of solution
What are the units of specific activity?
Units (of desired protein) per mg of protein total
What is the measure of purity of a protein?
The specific activity
How can proteins be precipitated?
1) Salt (NH4SO4) - best, most gentle
2) pH Extremes - can kill proteins
3) Heat - can kill proteins
How does Ion-Exchange Chromatography work?
- Beads in column have charged particles, either all positive or all negative
- Liquids w/ proteins going through will go through at different rates depending on charge or proteins
- Fractions will contain different proteins w/ different charges
How does Size-Exclusion Chromatography work? What is its other name?
Gel Filtration
- Bigger proteins go through column faster than small because small proteins get stuck in the porous polymer beads
How does Affinity Chromatography work?
- Polymer bound ligand (with affinity for protein of interest) is placed in column
- Protein mixture is poured through
- Unwanted proteins wash through column
- Protein of interest is eluted by ligand solution
How do you determine when the protein is pure?
- When further fractionation does not increase the specific activity
- Electrophoresis
What does electrophoresis take place in?
Polyacrylamide which allows the proteins to go through based on their molecular weights
What is the function of Sodium Dodecyl Sulfate (SDS)?
SDS binds to proteins in amounts proportional to their length, partially denatures the structures, as well as gives them all an overwhelming negative charge; this allows the proteins to move through proportional to their MW
How do proteins get visualized after they have gone through electrophoresis?
Coomassie blue dye which binds to the proteins but not to the gel
What trends should be seen in a Purification Table?
- Decrease in total protein (mg)
- Total enzyme (units) stays as high as possible
- Specific Activity (units/mg) increases
What kind of bonds make up the primary structure of proteins?
Covalent bonds: peptide bonds and disulfide bonds
How can the N-terminus be determined?
Using Sanger's method for direct protein sequencing;
- FDNB attacks the N-terminus and adds to end
- Strong Acid is added which removes the N-terminal A.A. with the FDNB leaving the remaining peptide chain
What are proteases? What are 4 we learned about?
Enzymes that catalyze the hydrolysis of peptide bonds
1) Trypsin**
2) Chymotrypsin**
3) Pepsin
4) Elastae
What does Trypsin do?
Protease which cleaves on the carboxyl side of Lysine or Arginine
What does Chymotrypsin do?
Protease which cleaves on the carboxyl side of Tyrosine, Tryptophan, and Phenylalanine
How does CNBr cleave peptides?
It hydrolyzes peptide bonds at the C-terminus of methionine residues
How are Disulfide Bonds cleaved?
1) Oxidation by Performic Acid


1) Reduction by Dithiothreitol
2) Carboxymethylation by Iodoacetate (important or the previous step will revert to a sulfide bond
What reactant is used in sequencing by the Edman Degradation?
1) Phenylisothiocyanate w/ OH-
2) H+
=> Phenylthiohydantoin Derivative (determine A.A. from this compound) and then continue on remaining peptide chain
How can large polypeptides be sequenced?
Breaking them down into smaller fragments and piecing it together
Specifically, what is the process for sequencing large polypeptides?
1) Hydrolyze to separate amino acids
2) Determine # of each A.A.
3) Conclude which protease(s) would be useful based on # of A.A. / # of breaks
4) Determine N-Terminus of Polypeptide (w/ Sanger Method: FDNB + HCl)
5) Reduce Disulfide Bonds
6) Cleave w/ proteases, establish sequence via overlaps
Why can't large polypeptides be sequenced solely by the Edman Degradation?
The longer the polypeptide chain the lower the overall accuracy of the sequencing; thus they must be broken down into smaller fractions.
How are Disulfide Bonds located (specifically which Cysteine residues are linked together)?
- Two samples, one cleave disulfide bonds, one leave them
- Cleave with protease
- Electrophoresis
- Compare fragments on a gel
- The run w/o disulfide bond cleavage should have larger bands; that w/ disulfide bond cleavage will have 2+ bands missing which is replaced by larger band
What is a Consensus Sequence?
A DNA or amino acid sequence consisting of the residues that most commonly occur at each position in a set of similar sequences
- use cool letter graph
- use symbols, [ ] = these A.A. could be present, x(#) = a lot of options could be here, { } = any AA except for these
The C-O bond is _____ than expected in peptide linkages.
The C-N bond is _____ than expected in peptide linkages.
C-O is longer than expected (not always "double" bond)
C-N is shorter than expected (not always "single" bond)
Because the peptide bond is not solely composed of a C=O and C-N bond... what effect does this have on the rotation of the peptide backbone?
There is not free rotation in the backbone of the peptide bond
Around which bonds is there free rotation in the peptide backbone?
N-C(alpha) and C(alpha)-C bonds have free rotation
Which rotation angle is referred to by ϕ (phi)?
C - Cα - N - C dihedral / torsion bond angle
(180° means N trans; 0° is cis)
Which rotation angle is referred to by ψ (psi)?
N - Cα - C - N dihedral / torsion bond angle
(180° means N trans; 0° is cis)
In an α-helix, are the R groups on the inside of the helix or outside?
Outside of helix
How far is one turn of the α-helix? How many amino acid residues is that?
0.54 nm
3.6 residues
In which direction do α-helices rotate?
Why are α-helices such stable conformations?
It makes optimal use of internal hydrogen bonds; the N-H is lined up with the C=O of the 4th A.A. on the N-terminal side
Why is Proline very rarely found in α-helices?
Its nitrogen atom is part of a rigid ring, making N-Cα bond rotation impossible, resulting in destabilizing kinks
What's the difference between Parallel and Anti-Parallel β sheets?
- Parallel - same amino-to-carboxyl orientations
- Anti-Parallel - opposite amino-to-carboxyl orientations
Are Parallel or Anti-Parallel β sheets more stable/common?
Anti-Parallel is more stable and more common (more direct H-bond alignment between N-H and C=O)
Why is silk so slippery?
Anti-parallel β sheets with small R groups (glycine and alanine)
How are the ends of anti-parallel β-sheets linked?
β Turns: 180 deg turn involving 4 A.A. residues w/ a H-bond between the 1st and 4th A.A. involved
How are the two types of β turns different?
- Type 1 - often includes Proline due to readily assumed cis configuration which is amenable to a tight turn
- Type 2 - includes glycine which is small and flexible (less steric hindrance)
What does a Ramachandran Plot show?
The possible and likelihood of different combinations of dihedral / torsion angles in proteins (ψ vs ϕ
- dark blue = most favored
- non blue = doesn't happen
Which type of dihedral band angle combination is suspected to be possible, but no one has ever found a long enough chain of it?
Left-handed α-helix
Which amino acid residue can fall outside of the Ramachandran Plot expected values?
Glycine due to its small H side-chain which allows it to take part in many conformations that are sterically forbidden for other a.a.
What is distinct about Fibrous proteins compared to Globular proteins?
- Fibrous usually have just one type of secondary structure with a simple tertiary structure
- Globular usually have multiple types of secondary structure
What are the typical functions of Fibrous proteins?
Provide support, shape, and external protection
What are the typical functions of Globular proteins?
Enzymes and regulatory functions
When two α-helices are twisted together what is it called? What is known about the handedness?
- Handedness is opposite that of the individual helices
In perms, what are the main steps?
1) Break disulfide bonds in hair with Thioglycolate
2) Curl hair
3) Oxidize sulfide bonds again; different linkages occur causing hair to curl
α-Keratin is an example of what kind of protein?
Fibrous protein - hair - made up of many superhelices - protofilaments - protofibril
What is the most abundant protein in the body?
What is the structure of collagen?
Single L-handed helix (not an α-helix) w/ 3 A.A. residues per turn; 3 α chains (separate polypeptides) are supertwisted about each other (right-handed)
Collagen is constructed of a repeating tripeptide made of what?
Gly-X-Y where X and Y = Proline or 4-Hydroxyproline
- Proline prefers C-endo conformation
- Y prefers to be in C-exo conformation which is less stable, but it is reinforced by the hydroxylation
How is Proline hydrolyzed to 4-hydroxyproline (the preferred conformation for c-exo in the Y position of the collagen tripeptide)?
Prolyl Hydroxylase hydrolyzes Proline; requires vitamin C (ascorbic acid)
What is it called when there are Vitamin C deficiencies? What happens?
Scurvy - collagen defects - gums bleed, sore joints, nose bleeds, die
How are collagen α chains cross-linked?
With unusual amino acid residues - dehydrohydroxylysinonorleucine - lysine residues covalently bonded to hylysine residues
What are two genetic defects involving collagen?
- Osteogenesis Imperfecta: affects bone formation in babies - Gly to Cis mutation (doesn't fit tightly)
- Ehlers-Danlos Syndrome: loose joints, soft skin, eye problems and bone deformations - Gly to Ser mutation
Why is gelatin (a protein) not a good source of protein?
Lacks many essential amino acids; primarily consists of glycine (35%), alanine (11%), and proline/hydroxyproline (21%)
What stabilizes the 3-D structure of globular proteins?
- Electrostatic Forces (ionic interactions / salt bridges)
- Hydrogen Bonds (small contribution, however unpaired donor or acceptor is detrimental to stability)
- Hydrophobic Forces (most important, increases entropy)
- Disulfide bonds
What are two pictorial ways to represent protein 3-D structure?
- Surface contour conformation (shows reaction sites)
- Ribbon representation (shows secondary structures)
What was the protein on which the preliminary understanding of tertiary structure was discovered? How?
Myoglobin by John Kendrew (1950s) with x-ray diffraction
What are the functions of myoglobin?
- Stores oxygen
- Facilitates oxygen diffusion in rapidly contracting muscle tissues
- Underwater mammals have a lot of this so they can stay under a long time
How does X-Ray diffraction give the structure of a protein?
- Protein is crystallized
- X-Ray beams diffracted through
- Pattern of spots is used to reconstruct the image of protein (e- density)
- Model structure into e- density map
- Answer of where atoms in protein are
How can scientists determine the structure of a protein?
- NMR (only for smaller molecules)
- X-Ray Diffraction
What did scientists learn about protein structure from myoglobin?
- Predicted secondary structure exists
- Very compact
- Hydrophobic residues buried
- Proline often found at bends
- All species similar; 3-D structure conserved over evolution
Are β sheets typically planar?
No, they tend to twist slightly in a right-handed fashion
How are two antiparallel β sheets connected?
β bends
What types of connections are more common between β strands?
Right-handed connections
What is a motif in protein structure?
A recognizable folding pattern involving two or more elements of secondary structure and the connections between them; may or may not be independently stable
What are some examples of motifs in protein structure?
- β barrel
- β-α-β loop --> α/β barrel (made up of many loops, much more stable than individual loop)
What is a domain in protein structure?
Part of a polypeptide chain that is independently stable; usually each domain has a specific, discrete function
How many domains are in a protein?
Depends on individual protein; some have multiple domains, some have just one
What are the 4 classes of motifs/folds?
1) All α
2) All β
3) α / β (interspersed)
4) α + β (segregated)
Serum albumin is in all α protein, what is it's function?
Carries many things, regulates osmotic pressure, found in bloodstream
Bacterioferritin (cytochorome b1) is an all α protein, what is its function?
E- carrier which binds Fe atom or cofactor
How is UDP N-acetylglucosamine acyltransferase's structure critical to its function?
It is an all β structure (parallel) which forms long deep pockets to synthesize long lipids in.
What is the purpose of Collagenase-3?
All β structure which looks like it has four bladed β propellers; used to get rid of collagen in places it does not belong
Alcohol dehydrogenase has which kind of fold structure?
The green fluorescent protein found in jellyfish for glowing has what kind of fold structure?
α +β
(β barrel w/ α parts)
What stabilizes quaternary structure?
The same factors which stabilized individual subunits (hydrophobic effects, hydrogen bonding, van der waals...)
How does a protein know which way to fold?
** it does not randomly sample all possible conformations **
- Formation of short stretches of secondary structure
- Formations of stable subdomains and domains
- Some proteins have loose "molten globule" intermediate
- Enzymes catalyze some steps (disulfide bond formation and proline isomerization)
** Chaperones **
What are chaperones and why are they useful?
Large protein complexes with huge chambers which allow proteins to fold up to proper structure
What ways can proteins be denatured?
- Heat (disrupts H-bonds and others)
- Urea (disrupts hydrophobic interactions)
- pH Extremes (disrupts ionic interactions)
- High [Salt] (disrupts ionic interactions)
- Detergents (disrupts hydrophobic interactions)
- HS-CH2-CH2-OH (mercaptoethanol, reduces disulfide bonds)
What does the term "denaturation" mean?
Loss of activity, not loss of ALL structure; just enough loss of structure so that protein loses its function
What lesson was learned from Anfinsen's experiment? How?
** Primary structure determines tertiary structure
- EXP: ribonuclease treated with urea and mercaptoethanol = denatured; when removed, the protein refolded precisely and regained activity = renaturation
What are Ligands?
Molecules that are bound reversibly to a protein; transient in nature
Where do ligands bind to proteins?
Binding sites which are complementary to the specific protein-ligand interaction
How flexible are proteins?
- Not rigid
- Undergo conformational changes (big or small)
- Breathing - molecular vibrations
What usually happens when a ligand binds to the specific binding site on a protein?
- Coupled with conformation change, causing an induced fit which is more complementary to the ligand
How are myoglobin and hemoglobin related/similar/different?
Myoglobin (Mb)
- 1 subunit, MW = 16,700
- Found in muscle for oxygen storage
- 4 subunits (α2β2), MW = 64,500 (4x myoglobin)
- Found in blood for oxygen transport
What does Oxygen bind to?
- Transition metals: Fe2+ is suitable for reversible binding
- Found in heme
Why do globins need heme / Fe2+ to bind oxygen?
No amino acid is suitable for reversible binding of Oxygen
Why must free iron in organisms be controlled / bound? How is this taken care of?
It can be very damaging to DNA and other macromolecules; It is sequestered, to make it less reactive, within a protein-bound prosthetic group called heme
When iron is sequestered in heme, what is the structure that directly surrounds the iron?
Porphyrin ring which prevents Fe2+ from changing to Fe3+ (does not bind oxygen)
Where is the heme located in heme-containing proteins?
Deep within the protein structure
The Fe2+ within the porphyrin ring has two open coordination bonds; what is attached to them?
1) Histidine residue - attached at N
2) Binding site for O2
Why is CO highly toxic to aerobic organisms?
It is coordinated to heme iron with greater affinity than O2; therefore O2 would be excluded and organism would die
What is the structure of myoglobin?
Single polypeptide with a heme molecule; made up of eight α-helical segments connected by bends
What is the equilibrium expression, which describes the reversible binding of a protein (P) to a ligand (L)?
P + L <-- --> PL
Ka = [PL] / [P][L] = 1/Kd
Ka = association constant [M^-1]
Kd = dissociation constant [M]
What does Ka (the association constant) measure?
The affinity of the ligand, L, for the protein, P.
What is the Binding Equilibrium equations?
Θ = Binding sites occupied / Total binding sites = [PL] / [PL]+[P]
Rearranged: Θ=Ka[L] / Ka[L]+1 = [L] / [L] + Kd
How does Kd related to binding?
Increase Kd= weaker binding
Decrease Kd= stronger binding
What is the relationship when half of the binding sites are occupied, Θ=1/2?
Dissociation constant Kd = concentration of the ligand
What is the specific equation for ligand/protein when Oxygen is the ligand?
Θ = [O2] / [O2]+[O2]_0.5
[O2]_0.5 = concentration at 1/2 saturated
What are the strong interactions holding hemoglobin together?
Strong interactions between "unlike" subunits (α to β, β to α); also hydrophobic interactions, H-bonds, and ionic pairs
Oxygen induces conformational changes in hemoglobin; what are the two states?
- T = "tense" - low oxygen affinity; predominates in absence of oxygen
- R = "relaxed" - high oxygen affinity
- oxygen binding triggers T to R transition
Why is a hyperbolic binding curve inappropriate for the binding of oxygen with myoglobin/protein that binds oxygen?
- If it was high-affinity, the protein would pick up oxygen well in the lungs (high pressure) but not release it in the tissues (low pressure)
- If it was low-affinity, it would be able to release the oxygen in the tissues, but wouldn't pick up much from the lungs
What is the appropriate binding curve for oxygen binding to proteins (myoglobin)? Why?
- A sigmoid cooperative binding curve
- Lower affinity for oxygen in lower pressures (tissues) allows it to release oxygen to tissues
- Higher affinity for oxygen in higher pressures (lungs) allows it to pick up oxygen in lungs
What does allosteric binding refer to?
The binding of one ligand affects the binding of others
What is the Hill equation?
log (Θ / 1-Θ) = n log [L] - log (Kd)
What is plotted on the coordinates of the Hill Plot?
y = log (Θ / 1-Θ)
x = log [L] ((log pO2))
b = - log (Kd)
m = n (Hill coefficient)
What does the slope of the Hill plot represent (n)?
When n = 1, ligand binding is not cooperative
When n>1, positive cooperation in ligand binding
There are two models of cooperative binding, what are they?
1) Symmetry model - subunits of a cooperatively binding protein are functionally identical; two conformations possible, all subunits undergo transition simultaneously
2) Sequential model - ligand binding can induce a change of conformation in individual subunits which increases the likelihood of nearby subunits transitioning
What can hemoglobin transport besides O2?
H+ and CO2 (inverse of O2 binding)
What state do H+ and CO2 prefer for hemoglobin?
T state = tense = low oxygen affinity
What is the Bohr effect?
- At low pH (high H+), high CO2, affinity of hemoglobin for oxygen is low and O2 is released in tissues
- At high pH (low H+), low CO2, affinity of hemoglobin for oxygen increases and more O2 is bound for transport
How does BPG affect oxygen binding?
Binding of BPG stabilizes the T (tense) state which decreases Oxygen binding... causing more oxygen to be released into the tissues
When does Sickle Cell Anemia occur?
Caused by mutation in hemoglobin (Glu to Val at pos. 6)
- Leads to fewer erythrocytes, unusual shape, weakness and pain
What are three principles of enzymatic catalysis?
1) Enzymes effect rates, not equilibria
2) Enzyme-transition state complementarity
3) Many parts of a substrate contribute
What are Ribozymes involved in?
- Mainly in RNA metabolism
- Exception: synthesis of proteins on ribosomes
What is a prosthetic group?
A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein.
Where do enzyme-catalyzed reactions take place?
Active Site
- analogous to ligand binding site (except that a reaction is facilitated)
- usually a cleft or pocket on the enzyme, lined w/ amino acid residues and cofactors positioned to optimally facilitate reaction
How is the standard free-energy change in biochemistry regulated?
pH = 7
What is binding energy used for?
- Entropy reduction - hold substrates in proper orientation to react
- Desolvation - replace H-bonds to H2O
- Strain - facilitates any geometric or electrostatic distortion required
- Induced Fit - bring reactive groups on enzyme into proper orientation for catalysis
What is heterolytic cleavage? Homolytic?
E- pair ends up entirely on one atom (most often)
E- pair split between two atoms to produce radicals (very reactive)
What is the most common reaction?
Proton transfer
What are cofactors?
Groups or molecules (other than AA residues) which are important to catalysis
i.e., metals (metalloenzymes), coenzymes (from vitamins)
What is an enzyme without a prosthetic group? With?
W/O: Apoenzyme
W/: Holenzyme - active
What are metals used for in enzymes?
Metals bind to substrate to orient them; mediate redox rxns; electrostatic shielding or stabilization of neg. charge
What does Mg2+ do for ATP?
Shields the negative charges; very highly charged is not ideal biologically, hard to go through membranes...
How do covalent catalysts occur?
- Formation of covalent complex w/ nucleophilic attack
- Catalyst must be linked to substrate transiently
What are some requirements of covalent catalysts?
- Catalyst (imidazole) must be better nucleophile than acyl acceptor (H2O)
- Intermediate must be more reactive than substrate
- Intermediate must be less stable than product