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238 Cards in this Set

  • Front
  • Back
1. What are Amino Acids?

Four things...
1. Weak acids/bases
2. Polar and ionizable
3. Except glycine all are chiral
4. Only L-amino acids are found in proteins
3. How is an amino acid residue formed
Remove water from amino acid
4. Describe an amino acid titration curve.

Three things...
1. Buffer region is the pKa +/- 1 pH unit

2. At pKa have 50% of each species

3. Titration curve can predict electric charge
6. What is the pI?

Three points...
1. Isoelectric point

2. pI = 1/2(pK1 + pK2)

3. The pH at which the solute has no NET electric charge
7. What is a zwitterion

Three things...
1. A dipolar ion

2. Can act as an acid (H+ donor) or base (H+ acceptor)

3. Does not have to be electrically neutral
8. What are the 4 classification of amino acid R groups?
1. Non-polar (hydrophobic)
2. Polar, Uncharged
3. Acidic
4. Basic
9. Describe the non-polar, aliphatic R Groups.

Three points...
1. Hydrophobic

2. Tend to cluster together in proteins

3. Stabilize structure through hydrophobic interactions
10. Describe glycine.

Five points...
1. R group is H
2. Achiral
3. Flexible b/c of small R
4. Highly conserved in proteins
5. Seen in loops joining alpha helices and beta sheets
11. Describe alanine.
1. CH3 is R & hydrophobic
2. One of the most common amino acids
3. Tend to be on surface and interior b/c of small size
4. Frequently used in mutagenesis studies
13. Describe alanine, valine, leucine, and isoleucine.

Three things...
1. Form protein core
2. Don't interact favorably with water
3. Different sizes and shapes permits them to cluster together and exclude water

*take advantage of hydrophobic interactions
14. Describe methionine

Three things...
1. One of 2 AA containing sulfur

2. Non-polar thioester group is NOT very reactive

3. Thioester is not at end
15. Describe proline.

Five points...
1. Unique b/c of ring structure
2.Ring structure isn't favorably for secondary protein structure
3. No amide H for H-bonding
4. Ring structure imposes rigid constraints on rotation
5. Highly conserved in proteins and doesn't easily handle substitution
16. Describe phenylalanine.

Four points...
1. Aromatic

2 Large, flat ring

3. Very non-polar

4. Chemically reactive extreme conditions (not biological)
17. Describe tyrosine.

Five things...
1. More polar than Phe
2. Can H-bond
3. Ionize at high pKa (10.5)
4. Can be phosphorylated
5. Absorbs light
18. Describe tryptophan

Four things...
1. Largest R group and most flourescent
2. Occurs less frequently (only have 1 or a few)
3. Tend to be highly conserved
4. Absorbs light at 280 nm
19. What doess the Lamber Beer Law tell?
Number of tryptophan and tryosine
20. Describe serine and threonine.

Four things...
1. Alcohols so soluble in water
2. Small, aliphatic
3. OH is not very reactive (pKa = 14)
4. Commonly phosphorylated
21. Describe cysteine

Four things...
1. Contains sulfur
2.Sulfhydroxyl group is reactive (pKa = 8.4)
3. Weakly polar
4. Important to stability by forming disulfide bonds
22. What are disulfide bonds in proteins?
Two cyteines oxidize to form a disulfide bond

*Contribute to protein stability
23. What did Christian Anfinsen do in his experiment with ribonuclease?

Three things...
1. Added urea and beta-mercapto-ethanol
2. Urea unfolded the protein so was inactive
3. Reduced disulfide bridges to yield cysteine residues
24. What were the conclusions of Anfinsen's experiment?

Three things...
1. Correct disulfide bonds stabilize native protein structure and are critical for activity

2. Primary structure or AA sequence dictates native tertiary structure

3. Denaturation and renaturation were reversible
25. What is the correct order of removing agents in Anfinsen's experiment to yield the correct disulfide bonds, and thus active form of the protein again?
1. Remove urea
2. Add trace of beta-mercapto-ethanol
3. Warm gently
26. What were Anfinsen's interpretation of the folding data?

Two parts....
1. Primary structure directs the protein folding

2. Free energy change drives renaturation
27. Describe lysine and arginine.

Four things...
1. Strongly polar
2. Strongly basic (high pKa, pH = 11-12)
3. Always positively charged at physiological pH
4. Usually found at surface of proteins where hydrated by water
28. Describe histidine.

Three things...
1. pH/pKa = 6
2. Greatest participant b/c pH is near physiological pH
3. Can play a key role in catalysis b/c of the ability for proton transfer
29. Describe aspartate and glutamate.

Four things...
1. Strongly polar, acidic residues
2. R group is deprotonated above pH 4 (pKa = 4)
3. R groups have a net negative charge at physiological pH
4. Tend to be at the surface of proteins
30. What are peptides?

Two points...
Two amino acids COVALENTLY linked through a condensation reaction

Peptides are named beginning with amino-terminal residue which is by convention placed at the left
31. Describe Protein Structure.

Four levels...
1. Primary
sequencof AA

2. Secondary
ordered structure; repeating conformation

3. Tertiary

4. Quartnery
association of several polypeptide chains
32. What are 3 principles of protein structure?
1. 3-D structure is determined by its AA sequence

2. Function of protein is dependent upon its 3-D structure

3. The 3-D structure of a protein is unique
33. What are the non-covalent interactions in proteins?

Three forces...
1. H-bonds
2. ionic
3. Hydrophobic interactions,

*cumulative effect provides stability
34. How does entropy play a role in protein folding?

Four points...
1. Entropy increase is main driving force for folding
2. Water molecules form solvation shell around unfolded polypeptide chain (entropy decrease)
3. As protein folds, hydrophobic residues are sequestered, solvation shell is disrupted
4. Entropy increases and number of H-bonds within the protein are maximized

*van der waals radius defines how close 2 atoms can be
35. How is the primary structure bonded?
1. Peptide bond is an amide bond, its planar and rigid
-have partial double bond characteristic

Carboxyl group of each AA to the alpha-amino group of the adjacent AA
36. What did Linus Pauling and Robert Corey study and find?
Studied peptide bond

Laid the foundation to our present day understanding of protein structure
37. What makes the peptide bond planar?

Three points...
1. Peptide Co - N is shorter than typical amine C-N but longer than C=N
2. There is resonance bwt carbonyl O and amid N resulting in small dipole
3. Peptide bond has 40% double bond character preventing free rotation
38. Describe a trans peptide bond.

Two points...
1. Carbonyl O is on opposite side of the peptide bond from amide H

2. Most are trans: favored energetically
39. Describe cis peptide bonds.

Two points...
1. Carbonyl O is on the same side as amide H

2. Only favored for proline and glycine
40. What is the secondary structure?

Three points...
1. Spatial arrangement of AA residues
2. Regular folding pattern

3. Examples: β conformation, α-helix, β turn
42. How did William Astbury contribute to secondary structure?
Evidence of secondary structures from x-ray diffraction studies on α-keratin
41. How did Pauling and Corey contribute to secondary structure?

Two points...
Predicted the existence of secondary structures

Proposed 2 classes of periodic structure: α-helix and β-sheet
43. Describe α-Helix

Five points....
1. R groups protrudes out
2. H-bonding of backbone
3. Stabilize helix through H-bonding and each peptide participates in H-bonding
4. Atoms in van der waals contact
5. Most common form in proteins is right-handed helix
44. Describe α-Helix Periodicity
3.6 aa per turn
45. How does amino acid sequence affects α-helix stability?

Five points...
1. electrostatic interactions (attraction or repulsion) bwt aa with charged R-group
2. bulkiness or shape of adjacent R group
3. interactions bwt an aa and the aa three to four residues away
4. occurence of proline or glycine
5. interaction bwt aa residues at ends of the helical segment and the electric dipole inherent to the α-helix
46. Why does proline affect the stability of alpha-helices?

Three reasons...
1. Prevents the amide N from participating in H-bonding
2. Steric hinderance b/c R group does not project outward
3. Produces a kink in the helix
47. What residues promote α-helix conformation?

Six of them...
1. Methionine
2. Glutamate
3. Glutamine
4. Alanine
5. Leucine
6. Histidine
48. What residues disrupt or are poor α-helix formers?

Eight of them....
1. Proline
2. Glycine
3. Tyrosine
4. Serine
5. Asparine
6. Threonine
7. Cysteine
8. Aspartate
50. Describe β-conformations.

Five points...
1. Has a zig zag backbone
2. H-bonding bwt adjacent segments of polypeptides
3. R groups protrude away and in opposite directions
4. Adjacent polypeptide chains in β-sheet can be parallel or antiparallel (having the same or opposite amino-to-carboxyl orientations)
5. Antiparallel have nice linear H bonds while parallel have distorted less stable H bond
51. Describe β-turns.

Two points...
1. Most proteins have compact globular shapes due to numerous reversals of direction of polypeptide chain

2. Loops connect structural elements

*small aa like glycine
52. Describe the tertiary structure.

Three points...
1. Overall folding of a single polypeptide chain into domains

2. Three-dimensional structure

3. Biologically active shape or native conformation
53. How are tertiary structures arranged? What gives them stability?
Stabilized by non-covalent interactions and disulfide bonds

*Hydrophobic interactions give stability to core

These interactions are driving force for folding
55. Describe Quaternary Structure or Multi-subunit Proteins.

Three points....
1. Organization of subunits to form oligomer

2. Arrangement of polypeptides to form native protein

3. Subunits usu assoc. non-covalently
56. Describe the process of protein folding.

Two points...
1. Protein folding cannot be a trial and error process

2. There must be folding pathways
57. What temperature are used when working with proteins during separation and purification?
Use low temperatures (4 C) and work rapidly to minimize degradation by proteases
58. When purifying and separating, you exploit differences in proteins based on....

Six properties
1. Solubility
2. Net charge
3. Regions of + and - charge
4. Size
5. Hydrophobic interactions and properties
6. Specific interactions with ligands
59. What are the four steps the in separation and purifying of proteins?
1. Homogenization
2. Differential centrifugation
3. Concentrate protein of interest
4. Later steps may include gel filtration and/or affinity chromatography
60. What is homogenization?
Done in buffer to obtain the homogenate or crude extract or cell extract

*ER is broken up
61. What are some choices of buffers used in homogenization based upon?

Five things...
1. Low ionic strength(help dissociate cytoskeleton)
2. Osmolarity (facilitates things falling apart)
3. Salts
4. pH
5. Additives
62. What happens in differential centrifugation?
Separate into:
1. 18K supernatant or cell extract
2. 18K pellet
63. What are two ways to concentrate protein of interest?
1. Precipitation methods

2. Ion exchange chromatography
64. What three things are used in precipitation methods?

How is it done?
1. ammonium salts: NH4(SO4)2
2. polyethyleneimine: PEI
3. polyethylene glycol: PEG

*obtain pellet, re-suspend in buffer and dialyzes to remove salt
65. What is ion exchange chromatography based upon?
Exploiting an unique feature
66. What resins used in ion exchange chromatography?

Four resins....
1. DEAE: positively charged anion exchanger
2. PC: negatively charged cation exchanger
3. S-Sepharose: negatively charged cation exchanger
4. Carboxymethyl: negatively charged cation exchanger
67. What is the process in ion exchange chromatography?

Six steps...
1. Add buffer on top to elute protein
2. Protein binds to functional group in resisn based on charge at pH
3. Elution of protein by displacement
4. Addition of counter ion binds resin more tightly than protein
5. First proteins to travel through don't interact with resin
6. Elute with salt to bump off bound protein
68. What is the purpose of SDS-Page?

What is it?

Four points...
1. Determine which part of fraction your protein is in
2. To follow protein of interest take sample of each fraction
3. Based on Mr
4. SDS binds most proteins
69. What is the process of SDS-Page?

Six points...
1. Mix 1:1 of fraction with sample buffer and heat
2. Protein unfolds to random coil/swamped with negative charge
3. All proteins have constant diameter, shape , and neg. charge
4. Stain with Commassie blue
5. Load into wells with buffer at top and bottom
6. Migrate based on molecular weight (Mr)
70. What are the results of SDS-Page?

Three points...
1. First lane is typically for standard of known Mr

2. Small migrate faster (lowest Mr)

3. Largest at top of gel (highest Mr)
71. What are some further protein purification techniques?

Three things...
1. Another type of ion exchange

2. Gel filtration

3. Affinity purification
72. What is gel filtration chromatography?

Three points...
1. Based on size AND shape

2. The resin synthesized with pores of specific size

3. Small proteins enter pores, large ones excluded

*large elute first since small interact with pore
73. What is affinity chromatography?

Two points
1. Most selective of purification strategies

2. Specific interactions with functional groups on resin
75. Describe the analysis of SDS-Page:
What does it tell you?

Two things...
1. Relative purity

2. Number different subunits
76. Analysis of Gel filtration: What does it tell you?
Size and shape-estimate of native Mr

*remember may not be accurate (b/c of shape rod & globular would elute the same)
77. Analysis of native gel electrophoresis: What does it tell you?
Size and Charge
78. What happens in isoelectric focusing & 2-D electrophoresis?

Four things...
1. Proteins migrate in electric field based on charge
2. Stop at pI b/c electrically neutral
3. Next run gel under reducing conditions (SDS-page, 2-D electrophoresis)
4. Migrate based on Mr
79. What is seen in a 2-dimensional gel?

What do these represent?
1. Horizontal separation: differences in pI

2. Vertical separations: differences in Mr
80. What are the two dimension of of 2-D gel electrophoresis?
1. Migration based on pI:

2. Migration based on Mr (smallest travels furthest on gel)
81. What was the first protein sequence and who did it?
First protein sequenced was insulin

Sanger sequenced it
82. What are two way to tell amino acid composition?
1. Acid hydrolysis

2. Edman degradation
83. What does acid hydrolysis tell about the protein?

What is used in it?
1. Tells identity and # of aa

2. No sequence info

3. HCl: cleaves peptide bonds
84. What four amino acids are destroyed in acid hydrolysis?
1. Tryptophan
2. Serine
3. Threonine
4. Tyrosine
86. Describe Edman degradation?

Two points...
1. Sequential removal and identification of 1 residue at a time from the N-terminus

2. Some amino acids are lost (98% efficiency)

*Cystines must be reduced to cysteine
87. How is the N-terminus amino acid identified in Edman degradation?
Identify the amino-terminal amino acid residue using 1-fluoro-2,4-dinitrobenzene (FDNB)

*Dansyl chloride is also used
88. How do you sequencing larger proteins?

Two things....
1. Cleave polypeptide chain with proteases

2. Disulfide bonds are reduced
89. What are some reagents used for fragmenting polypeptide chains?

Four of them...
1. Trypsin (most common)
2. Chymotrypsin
3. Straphylococcus aureus V8 protease
4. Cyanogen bromide
90. Where does Tyrpsin cleave?
Lysine and Arginine
91. Where does Chymotrypsin cleave?
Phenylalanine, Tryptophan, and Tyrosine

*not as specific
92. How does Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry work?

Three points...
1. Proteins are placed in light-absorbing matrix
2. Pulse with laser light to ionize
3. Desorption from the matrix into vacuum system
93. How does Electrospray Ionization Mass Spectrometry work?

Three points
1. Protein solution is forced through a charged needle at high electric potential
2. Solution is dispersed into mist of charged micro-droplets
3. Solvent rapidly evaporates and multiply charged ions are introduced nondestructively into gas phase
94. What are three methods for determining the 3-D structure of a protein?
1. X-ray crystallography

2. NMR

3. Electron microscopy
95 Describe X-ray crystallography.

Three points...
1. Info about the spacing of atoms in the crystal lattice
2. Protein must be able to be crystallized
3. Won't get crystal if solution is not homogenize
97. What are three advantage of NMR?
1. Can analyze protein in solm
2. Don't require crystallization
3. Get data about protein folding and interactions with other molecules
96. What NMR (nuclear magnetic resolution)?

Three points...
1. Info about dynamics

2. Have crystal structure also

3. Limited to < 30,000 MW
98. Describe Cryo-Electron microscopy?
Get structural info about protein complexes

*Cannot get this with other two methods
99. What is some general information about Myoglobin and Hemoglobin?

Three points...
1. Important O2 binding proteins

2. Myoglobin: muscle/tissue

3. Hemoglobin: blood
100. What are three modulators or effectors of Hb?

Where do they bind?
1. Protons (H+)
2. CO2
3. BPG:2,3-bisphosphoglycerate

Bind to sites separate from where O2 binds
101. Describe the structure of Mb.

Four points....
1. Single polypeptide
2. Single heme group
3. Bind 1 O2
4. Mostly alpha-helical
102. Describe the structure of Hb.

Five points
1. Four polypeptide chains
2. Similar aa sequence and tertiary structure to Mb
3. Four heme groups (1/chain)
4. Bind 4 O2
5. Subunits associate
103 What makes Mb stable?

Three points....
1. Derived from hydrophobic interactions
2. Eluded water from interior of protein (were O2 binds)
3. All but 2 of polar groups are on the surface
104.Describe the Heme group in Mb.

Three points....
1. Sequestered in a hydrophobic environment
2. Oxygen binding depends on Fe2+ (ferrous form)
3. Heme must be protected from water
105. What is the Heme group?

Two points...
1. Fe2+ in center

2. Prefers to be bound to 6 ligands
106. What are two important points about oxygen binding to Mb Heme?
1. Protect O2 from water with Val, Phe, and His

2. CO and NO bind to heme Fe2+ with higher affinity than O2
107. Why can CO and NO sneak in the Mb heme?
1. Transient access

2. Transient cavities
108. Describe the four heme groups of Hb.

Two points...
1. One per polypeptide chain

2. Each partially buried in hydrophobic pocket
109. How do Mb and Hb differ in oxygen binding?
Difference in oxygen binding is due to 4 heme in Hb (positive co-operation), NOT quaternary structure
110. What is the quaternary structure of Hb?

Three points...
1. Strong interactions bwt α1β1 and α2β2
2. Hydrophobic interactions predominate and H-bonds
3. Oxygen is mostly on β subunits
111. What happens when oxygen binds to Hb?
Pocket between β subunits narrows

*This pocket is BPG binding site
112. Describe oxygen binding to Mb.

Four points...
1. One binding site for oxygen
2. Binding is hyperbolic
3. No co-operativity
4. Mb has high affinity for O2 even at low [O2]

* measure partial pressure of oxygen
113. What is K1/2 or P50?
Concentration at which 50% of sites are occupied w/ O2

*When P50 is close to zero there is tight binding
114. What is the association constant?
Ka: forming complex

*higher Ka, higher affinity for ligand (tighter binding)
115. What is the dissociation constant?

*Higher Kd, lower affinity (weaker binding)
116. What is the fractional binding θ?
θ= binding site occupied/total

θ=[PL]/[PL]+[P] OR

117. What does it mean when [L]=Kd?
Point at which 50% is found occupied w/ O2

118. Describe xygen binding to Hb: graphical.

Four points...
1. Hb binding is sigmoidal
2. Indicative of cooperativity
3. Higher[O2] required for 1st O2 to bind
4. Affinity for O2 increases 500-fold after first 02 is bound
119. What is the physiological relevance of P50 for Mb and Hb?

Two points...
1. P50 for Mb quite tight SO Mb stores O2

2. P50 for Hb much weaker SO Hb transports O2
120. What does it mean that Hb oxygen binding is not hyperbolic?

Three points...
1. Positive co-operativity
2. Binding data is sigmoidal
3. Data better fit to the Hill equation
121. what does the "n" coefficient in the hill equation tell?

What's the "n" for Mb and Hb?
Degree of co-operativity (how many subunits are cooperative)

Mb: n = 1
Hb: n = 2.8-3 (almost never see 4 even though have 4 subunits)
122. Describe 2,3-biphosphoglycerate (BPG)

Three points...
1. Low Mr

2. Negative charge

3. One BPG per Hb
123. Where does BPG bind?

Three points...
1. Binds the deoxyHb
2. BPG binding site is in cavity
3. BPG-Hb interactions are all on β subunits
124. How does BPG binding affect Hb?
1. BPG stabilizes the deoxyhemoglobin state, which facilitates release of O2

2.BPG interacts with positively charged aa on β-subunits
126. How is the regulation of PBG physiological significant?

Three points...
1. Oxygenated Hb travels to tissues, begins to release O2
2. BPG binds which weakens binding further facilliating O2 release
3. higher[BPG], weaker O2 affinity
127. Describe fetal Hb.
Which amino acid is replaced?
What is the significance of this replacement?
1. Has serine rather than β-His 143
2. Loss of 2 positive charges in HbF makes it bind BPG less tightly
3. HbF binds O2 more tightly than HbA (since it binds BPG weaker)
*α2ϒ2 rather than α2β2 of HbA
128. What is the significance of tighter O2 binding in HbF?

Two points...
1. Facilliates O2 transfer to fetus

2. Guarantees that fetus is taken of first if O2 supply is low
129. How is H+ and CO2 transported by Hb?
Hb carries CO2 and H+ from tissue to lungs and kidneys for excretion

*catalyzed by carbonic anhydrase
1. What are the four non-covalent interactions?
1. Hydrogen bonds (between polar groups)
2. Electrostatic interactions (ionic bonds)
3. Hydrophobic interactions
4. Van der Waals
2. What are some characteristics of non-covalent bonds?

Two points...
1. Individually weak, cumulative effect confers stability

2. Small energy investment to break one bond (weak)

*For macromolecules most stable structure is usually that in which weak-bonding possibilities are maximized
3. What is configuration

Three points...
1. Fixed spatial arrangement of atoms
2. Conferred by
double bond and chiral center
3. Configuration cannot be changed w/o breaking one or more covalent bonds
4. What is conformation?
Spatial arrangement of groups that are free to assume different positions in space because of rotation about single bonds

*two important configurations
1. staggered: most stable
2. eclipsed: least stable
5. What are the three laws of thermodynamics?
1. Energy is conserved
2. Entropy tends to increase
3. Entropy of any crystalline, perfectly ordered substance, must approach zero as temperature approached 0K. At T= 0K entropy is exactly zero
6. What is Free Energy (G):
Amount of energy available to do work

ΔG = G products – G reactants
7. What is Entropy (S)
A measure of energy due to randomness of disorder

*products more disordered than reactants, gain in entropy (+S)
8. What is Enthalpy (H)?
Heat content of reacting system

Exergonic: release of free energy; neg. ΔG; spontaneous

Endergonic: needs investment of energy; pos. ΔG, non-spontaneous
9. What is the equilibrium effect?
Reactions can be driven in part by concentration of reactants and products

*at equilibrium no net change in [Reactant] and [Product]; no driving force
10. Describe the equilibrium constant Keq

Three points...
1. Large Keq means reaction proceeds till almost all of reactants become products
2. Keq > 1 ΔG is large and neg.
3. Keq < 1 ΔG is large and pos.

*At equilibrium: ΔG°’ = -RT lnKeq
11. What is the difference between ΔG°’ & ΔG?
ΔG°’(standard free energy chang): constant based on standard conditions

ΔG: actual free energy change for reaction and is based on actual experimental conditions
12. Describe the typical prokaryote.

Four points...
1. Unicellular
2. DNA in nucleoid, no nucleus
3. No membrane bound intracellular organelles
4. No mitochondria
13. Describe the typical eukaryotes (animal or plant cell)

Five points...
1. Typically multicellular
2. Membrane-enclosed nucleus
3. Membrane bound organelles
4. Nucleus with chromosomes
5. Enzymes and specialized functions segregated
14. What are some physical properties of water?

Two points....
1. Interacts with polar and non-polar substances

2. Has high melting pt, boiling pt, heat of vaporization, and surface tension

•these are result of hydrogen bonding that gives liquid water great internal cohesion
15. Describe the structure of water?

Five points...
1. H-bonding
2. Nonlinear H-O-H bond angles
3. Two other orbitals: unshared electron pair
4. O is more electronegative
5. Uneven distribution of charge(electric dipole)
16. Describe Hydrogen Bonding and Water.

Three points...
1. A H-bond is transient (“flickering clusters”)
2. At any one time, liquid water form H-bonds with 3.4 other water molecules
3. one water molecule can form up to 4 H-bonds
17. Why is water cohesive?
Cohesive because of the large number of H-bonds between molecules
18. What happens when ice melts?

Four beefy things...
1. Break H-bonds (accounts for high melting pt)
2. Entropy increases (liq water molecules less ordered)
3. Take heat from surroundings
4. Entropy drives reactions
19. What are the properties of ice?

Three chunks...
1. 4 H-bonds

2. More open lattice

3. Density of ice < liq water
20. Describe the properties of liquid Water.

Three things...
1. 3.4 H-bonds

2. More dense than ice

3. Molecules can pack closer together
21. How does water act as a solvent?

Four points...
1. Interacts with ionic and other polar substances
2. H acceptor: N, O, etc.
3. H donor another electronegative atom
4. C-H bonds do not participate in H-bonding
22. How do non-polar gases interact with water?

Two things...
1. Nonpolar gases are not very soluble in water

2. Don’t pass through membrane easily

*ex. CO2, O2, N2
23. What does it mean when we say fatty acids are amphipathic?
They have non-polar regions that cluster together to present the smallest hydrophobic area to aqueous solvent, while polar regions are arranged to maximize interaction with solvent
24. What happens when fatty acids are put in water?

Three step process...
1. Dispersion of lipids in water
-each lipid molecule forces surrounding water molecule to become highly ordered
2. Clusters of lipid molecules
-only lipid portions at edgy of cluster force ordering of water, few water molecules ordered, entropy increase
3. Micelles
-hydrophobic groups sequestered from water, ordered shell of water molecules is minimized, further entropy increase
25. What are hydrophobic interactions?
Forces that hold the non-polar domains of the molecule together

*minimize number of ordered water molecules required to surround hydrophobic domain by sequestering hydrophobic region
26. What are Van Der Waals interactions?
When bring 2 uncharged atoms close together, their surrounding e- clouds influence each other

*Create a transient dipole
27. What is the Van der Waals radius?

Two points...
1. A point where attractive force = repulsive force

2. How close atom will allow another atom to approach; if go too close atoms become repulsed
28. What is the ionization of water?

Does this happen easily?
1. Form H ion (H+) and hydroxide ion (OH-)

2. Water doesn't readily dissociate (due to H-bonding)
3. H+ is too transient to exist in solution (immed hydrated to hydronium ion (H3O+)
30. What is the equilibrium constant for water?
Kw = 1 x 10^-14
Kw(ion product of water) = (55.5 M)(Keq)
Kw = 1 x10-14 M2 = [H+][OH-]
-basis for pH scale
-pure water is electrically neutral (pH = 7)
[H+] = [OH-]
Kw = [H+]2
-both [OH-] and [H+] equal 1 x10-7 M
-if increase [H+], then [OH-] must decrease and vice versa
31. What is the pH scale?
pH = -log[H+]

-expresses [H+]
-neutral pH = 7
32. Describe Weak Acids and Bases
1. Most biological molecules are not strong acids or bases so don’t completely dissociate in water

2. Stronger the acid, greater the tendency to lose proton
Ka (dissociation constant)
Keq = [H+][A-] / [HA] = Ka
-stronger acids have larger Ka and weak acid has smaller Ka
pKa = -logKa
-logarithmic scale
-express dissociation constant
Henderson-Hasselbalch Equation
pH = pKa + log[CA]/[CB]
- greater [A-], higher pH
- when [weak acid] = [weak base], pH = pKa b/c log 1= 0
Titration Curves
-reveal pKa of weak acid and info about the behavior of amino acids
-add little base at low pH = sharp increase
-in middle pH, add base and slow rise (level)
-accumulate more conjugate base (anion) but still have acid
-when have little left of acid, sharp shoot up because it will titrate easily
pKa and titration curves
-the midpoint [acid] = [conjugate base]
- pH = pKa
• +/- 1 pH of pKa is buffering region
• +/- 2 pH, no buffering capability
-low pH, expect 100% of acid as acid
-high pH, expect 100% of dissociation of acid
What does it mean to not have buffering capability?
-not enough to equalize other specie ( only have 99% of one specie)
Phosphate System
-multiple groups dissociate
1. have three midpoints
2. as hit each groups buffering region, the previous group is gone
3. the last group (one with highest pKa) won’t dissociate until groups before it have completely dissociated
Bicarbonate Buffer System
• pH of bicarbonate system depends on [H2CO3] and [HCO3-]
• [H2CO3] in turn depends on concentration of dissolved CO2 which in turn depends on concentration of CO2 in gas phase
• CO2 is not readily soluble in aqueous solutions
1. 20% transported directly by hemoglobin
2. 80% transported by bicarbonate system
Hemoglobin: transport H+ and CO2
-carries CO2 and H+ from tissues to lungs and kidneys for excretion
-rxn catalyzed by carbonic anhydrase
-CO2 is not very soluble, hydration of CO2 increases H+ -> decrease in pH at tissues
-deoxyHb binds 2 H+ directly to each α-subunit N-terminus
-Hb can also bind CO2 directly to α-amino residues of each chain
-these are covalent rxns
Binding of O2 and H+ is antagonistic
-bind at different sites
-deoxyHb binds 2H+ directly to each α-subunit N-terminus
-high [H+] protonates other aa also (not just two binding sites)
DeoxyHb form and O2 and H+ binding
-deoxy form is when BPG is bound
-DeoxyHb at His 146 of β-subunits
1. His146H+ ••• Asp94- stabilizes deoxy form
-O2 binding drives conformational changes that destabilize
His146H+ ••• Asp94- interaction
Bohr Effect
-effect of CO2 and pH on O2 binding
-decrease in pH weakens affinity for O2
-in deoxyHb state, pH is low, aa more likely to be protonated (lower affinity for O2)
What is the significance of Mb being tissue specific?
-binds O2 tightly
Physiological Significance of Allosteric Regulation
1. oxygenated Hb travels to tissues, begins to release O2, BPG binds which weakens binding further facilitating O2 release
2. exercise, pH decreases (more acidic), weakens affinity for O2, release to muscles and tissues b/c in need
3. increase in CO2 and H+ results in decrease in pH and weakening of Hb's affinity for O2 (Bohr Effect)
Physiological Significance of Hb quaternary structure
-Hb is regulated first by positive cooperativity and then by 3 allosteric regulators
-at lungs, Hb is fully charged w/ O2
-at the tissue/muscle, Hb's affinity for O2 is less so can release O2
Sickle-Cel Anemia and Sickle-cell trait (HbS)
-genetic disease either homozygous or heterozygous
-single aa change: Glu6Val substitution on β chain
-change from neg aa to hydrophobic aa changing shape (sickling affect)
HbS Gluβ6Val
-Gluβ6 is on the outer surface
-Valβ6 switch results in sticky hydrophobic patch
-abnormal quaternary assoc of Hb subunits
-conformation change only occurs in deoxyHbS state
What happens when [O2] is below critical level and have HbS
-HbS becomes deoxygenated, insoluble and forms bundles of tubular fibers
-insoluble fibers of deoxyHbS deform RBC's
-serious consequences at small blood capillaries (blockag, tissue atrophy)
-if keep blood well oxygenated, less likely to have problem
Summary of Mb and Hb
Mb Hb
polypeptide 1 4
quaternary struc. no yes
heme groups 1 4
O2 that can bind 1 4
cooperative binding no yes
Hill coefficient (n) 1 2.8-3
BPG binding site none 1
allosteric reg. no H+,
Breaking disulfide bonds in proteins
-oxidation of a cystine residue with performinc acid produces 2 cysteic residues
-reduction to for Cys residue must be follow by further modification of the reactive -SH groups to prevent re-formation of the disulfide bond
Globular Proteins
-polypeptide chains folded into a spherical or globular shape
-often contain several types of secondary structure
Fibrous Proteins
-polypeptide chains arranged in long strands or sheets
-usually consist largely of a single type of secondary structure
-insoluble in water
-high concentration of hydrophobic aa residues both in the interior and on the surface
-intermediat filament protein (family)
-supertwisted coiled coil (two strands oriented in parallel are wrapped around one another)
-supertwisting amplifies the strength of the overall structure
-helical path of the supertwist is left handed
-intertwining of two α-helical polypeptides is exampple of quaternary structure
Strength of fibrous proteins
-enhanced by covalent cross-links bwt polypeptide changes w/in the multihelical "ropes" and bwt adjacent chains in the supromolecular assembly
-secondary structure distinct from α helix
-left handed and has 3 aa residues per turn
-three separate polypeptides (α chains) are supertwisted about each other
-superhelical twisting is right handed in collagen
-aa sequence in collagen is tripeptide unit: Gly-X-Y where X is often Pro and Y is often 4-Hyp
-Gly is at tight junction where 3 chains contact b/c of small size
-center of 3-stranded superhelix is not hollow (tightly packed)
-Gly cannot be replaced by another aa w/o substantial deleterious effects on collagen structure
Silk Fibroin
-rich in Ala and Gly permitting a close packing of β sheets and an interlocking arrangement of R groups
-silk does not stretch b/c β conformation is already highly extended
-structure is flexible b/c sheets are held together by numerous weak interactions rather than by covalent bonds (such as disulfide bonds in α-keratin)
Molecular Chaperones
-specialize proteins that facilitate folding for many proteins
-interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur
R state and T state
-O2 has higher affinity for Hb in the R state
-O2 binding stabilizes the R state
-T state is predominate conformation of deoxyHb b/c T state is more stable
-T state denotes "tense" and is stabilized by greater # of ion pairs
-R state is "relaxed"
-binding of O2 to Hb subunit in T state triggers change in conformation to R state
Tissue concentration of CO2 and O2
-CO2 is high in peripheral tissue, some CO2 binds to Hb and affinity for O2 decreases causing its release
-conversely, when Hb reaches lungs high O2 concentration promotes binding of O2 and release of CO2
R group pKa's
tyrosine: 10
cysteine: 8.18
Lysine: 10.53
histidine: 6
arginine: 12.5
aspartate: 3.65
glutamate: 4.25
AA abbreviation
Glycine: gly
Alanine: ala
Proline: pro
Valine: val
Leucine: leu
Isoleucine: lle
Methionine: met
Phenylalanine: phe
Tyrosine: tyr
Tryptophand: trp
Serine: ser
Threonine: thr
Cysteine: cys
Asparagine: asn
Glutamine: gln
Lysine: lys
Histidine: his
Arginine: arg
Aspartate: asp
Glutamate: glu
Symmetry Model Assumption
1. allosteric protein is an oligomer of symmetrically related or functionally identical subunits (either in one conformation or another)
2. each subunit can exist in 2 conformations:
-R or high affinity
-T or low affinity conformation (more stable absence L)
3.the ligand can bind to a subunit in either conformation
*only the conformation change alters affinity for the ligand
4. molecular symmetry of the protein is conserved during the conformational change
-all subunits switch simultaneously
-oligomers only contain all R- or all T- state subunits
Symmetry/Concerted Model
-all or nothing of one state
-ligand can bind to either conformation
-it is the conformational change (T -> R) that alters affinity
-symmetry is conserved; therefore, the subunits all R- or all T- state (all switch at one time)
Limitations of Symmetry/Concerted Model
-idea of oligomeric symmetry being perfectly preserved only accounts for positive cooperativity
Sequential Model
-ligand binding induces conformational change in subunit
-each ligand generates conformational change
-this conformational change affects neighboring subunits such that second ligand binding even more likely
-model proposes that there are intermediate states
-in reality both models are applicable
Properties of Enzymes
-specific and efficient catalysts
-most are proteins
-catalytic activity is dependent upon native protein conformation (if unfold protein, lose enz. function)
-primary through quaternary structure of protein enzyme are essential to catalytic activity
-additional factors (cofactors, coenzymes) may be required for activity
-coenzymes or cofactors are transient carriers of specific functional groups in active site
Enzyme Modification
-enzymes can be modified by posttranslational modification
Enzyme Active Site
-where chemistry happens
-site of catalysis
-site where substrate binds and is converted to product
-specific for substrate and cofactors/ coenzymes
-generally sequestered from soln (buried in interior)
-always a few polar, ionizable (R group) residues
-frequently a few water molecules
Alloster Site or Modulator Site
-separate site from active site
-allosteric modulators or effectors affect catalysis directly or indirectly
-can up regulate or down regulate activity
-on surface (charged residues)
Enzyme Specificity
-substrate,cofactors,coenzyme specificity
-reaction specificity
-enz provides specific environment w/in which a given rxn can occur more rapidly
Active Site Principles
-aa residues around active site determing specificity for binding
-sequestered by hydrophobic residues
-removed from water
-not opened on surface (in cavity)
Proximity Effect
-binding of substrate(s) <=> ES complex increases probability of ES <=> P + E in comparison to E + S in soln
What does the active site of the enzyme bind?
-active site binds the transition state of the substrate more tightly than the ground state of S
Transistion State of S‡
-an activated form of the substrate
-cannot be isolated experimentally (transient)
-not an intermediate
-weak interactions bwt the S‡ and active site optimized
-decay to S or P is equally probable
-typically noncovalent bonds
-substrate -enzyme complex
-can be isolated
-not transient
-finite chemical life
Active Site Amino Acids Participates in Enzyme Catalysis
-direct interaction in catalysis, often as proton donors or acceptors
-stabilization of substrate or substrate intermediate
-Glu, Asp, Lys, Arg, Cys, His, Ser, Tyr
What catalytic groups contribute to catalysis in enzyme mediated reactions?
-acid-base catalysis
-covalent catalysis
-metal ion catalysis
Acid-base catalysis
-rxn accelerated by catalytic transfer of proton
-R group active site can accept and transfer protons which provides rate enhancement
-80% use
Covalent Catalysis
-transfer of functional groups from S ->E, S2, coenzyme
-covalent interactions bwt E and S lower Ea by providing alternate, lower-NRG pathway
Rate of reaction and pH
-enzymes are active in narrow pH range (5-9) b/c of physiological pH
-if exceed pH range, protein degradation
pH optimum
-base on ionizable aa residues at the active site
-important for catalytic rxn
-pH range over which an enzyme undegoes changes in activity can provide a clue to the type of aa residue involved
-pKa of aa side chaing can be altered though due to close packed environment of protein
pH profile
-bell-shaped: 2 ionizable groups
-sigmoidal: 1 ionizable group
-can be more complicated though
Pepsin and Glucose 6-phosphatase pH
-pepsin has pH optimum of 1.6, disgestive enzyme in stomach
(pH is indicative of Asp or Glu involved)
-glucose 6-phosphatase has 2 ionizable groups critical for optimal activity
*at max though, one group is protonated and the other is deprontonated
Transition State Theory for Enzyme Catalysis
-rate of rxn is dependent upon the reacting species colliding and forming transisiton state
Free energy and standard free energy change
1. Free Energy (G): energy available to do work
2. Overall stander free energy change (∆G'°): linked with equilibria (S ->P)
Reaction Coordinate Diagram
-"energetic hill" is energetic barrier bwt S and P
-at TS, fleeting molecular moment at which decay to either S or P is equally possible
-enzymes don't affect equilibria just lower energy required to reach TS
"energetic hill"
energy required for alignment of reaction groups involving the breaking of bonds, bond arrangments, formation of transient unstable new bonds
Rules of Enzymes
1. lower activation energies to increase rates
2. substrate loses entropy when binds E (increase entropy for water b/c strip them away)
3. some interactions are formed upon S binding to E
4. full complement of possible weak interactions bwt S and E are formed only when S reaches the TS
5. free energy from substrate binding partially offsets energy required to reach TS
How does binding substrate give energy?
-formation of each weak interaction in ES complex is accompanied by release of small amount of free energy that provides a degree of stability to reaction (binding energy)
Two fundatmental principles of enzymes
1. catalytic power is derived from free energy released in forming multiple weak bonds bwt S and E
-binding energy provides specificity as well as catalysis
2. weak interactions are optimized in the TS
Two models for enzyme-substrate specificity
1. Lock and Key: active site is complementary to substrate
2. acitive site is complementary to transition state of substrate
Lock and Key Model
-no catalysis if exactly complementary b/c energitically unfavorable to break ES complex
-very stable ES
Enzyme complementary to TS model
-optimal interactions occurs at TS
-some interaction when S first binds, but binding releases energy for step by step binding to reach TS
Experimental Evidence for active site complementary to TS
1.Transition state analogs: observation that many inhibitors bound E more tightly than S
2. Catalytic Antibodies: direct evidence
Catalytic Antibodies
hypothesis: If ts analog can be designed for the rx S->P than an antibody to the analog may catalyze the rxn
Catalytic Antibodies: Exp
-direct evidence
1. antibodies don't do catalysis
2. took inhibitors that bound E tight and made antibodies of them (anti-body analogs)
3. if active site of E is complementary to TS of S then antibody analogs should do catalysis
4. did catalyze the enzymatic reaction
Enzyme and Contributions of bindig energy for catalysis
1. entropy reduction: E can hold substrate(s) in the proper orientation to react (constraint of motion)
2. formation of weak bonds bwt ES result in desolvaion of S (replacement of water molecules)
3. binding NRG compensates for any distortion S must undergo for rxn
4. E also undergoes change conformation so can optimize noncovalent interactions with S
How can we understand how an enzyme functions in vivo?
-define its mechanism and the structural transitions that occur
-always begin at steady state and then can look at other parameters
-put together mechanism to understand structural changes
How does an enzyme progress through a catalytic reaction?
-at start all active sites are able to bind
-free active sites decrease
-then at end all are free again
-regenerate original E
What did Brown concluded about enzymes through hydroylsis of sucrose?
-substrate binding at [S] became faster than formation of P therefore E sites must be satuarted
-rate was proportional to [S] at low [sucrose] (initial region)
-at high [sucrose] > [E] the rate was independent of [E] (implies E sites must be satuarated)
Michaelis and Menten concept of equilibrium
-at initial perod of the reaction
k-1 >> k+2, the first step achieves equilibrium b/c ES breaks down to E +S more often than it breaks down to form P
-thus k+2 is rate-limiting step
-overall rate is proportional to [ES] b/c it reacts in sencond step which is rate-determining
ks= (k-1)/(k+1) = [E][S]/[ES]
-ks: apparent dissociation constant of ES (takes into account all steps)
-originally proposed by michaelis and menten
-now called Michaelis constant (km)
"steady state approximation"
[S] >>>>> [E]
-after a few turnovers, [ES] is constant because [S] is so high , as soon as E is free it's grabbed by S
-net change in [ES]=0
steady state
-formation of ES = disappearance of ES
-ES is constant
-ES disappeares by going forward or S can fall off active site
Assumptions in kinetics
1. binding of S by E results in ES complex
2. if E is not saturated w/ S, small amound of P is formed. [P] is extremely low and k-2 is negligible (initial stage)
3. ES cannot be measured easily
4. at steady state, [S] is very very high relative to [E]
2. What are the pKa's of the carboxyl and amino groups in amino acids?
1. alpha carboxyl pKa = 2

2. alpha amino pKa = 9
5. Where is the worst buffer region?
Worst buffer region is where have steep increase because only one species is present
12. What is mutagensis?

How is alanine used in it?
1. Substitute one AA for another to find out if function is loss

2. Substitute AA with alanine

3. Small size typically does not change the protein conformation
32. What are the most important forces in stabilizing the native conformation(s) of a protein?
The non-covalent interactions

*some disulfide bridges are essential
49. Why are these things bad in alpha-helices?

Three reasons...
1. Contain a H-bond donor or acceptor in close proximity to main chain
2. Multiple glu or asp in sequence (repulsion)
3. Multiple lys or arg in sequence (repulsion)
54. How are amino acids arranged in the tertiary structure?

Three points...
1. Hydrophobic aa side chains are sequestered to the interior
2. Charge aa are almost always on the surface
3. Uncharged polar residues can occur at the surface or the interior
74. What is the process of affinity chromatography?

Four steps...
1. Add protein mix to column containing a polymer-bound ligand specific for protein of interest
2. Unwanted proteins are washed through column (don't bind with ligand)
3. Add solution of ligand
4. Protein of interest is eluted by ligand solution
85. What amino acids cannot be distinguished from each other in acid hydrolysis?
1. Cannot distinguish Glutamate from Glutamine

2. Cannot distinguish Asparate from Asparnine
125. How does O2 affect BPG binding?
When oxygen binds, the binding site for BPG is not accessible

*Closure of BPG binding pocket
29. What does the ionization of water allow it to act as?
Can be either an acid or a base b/c it can accept or donate proteins