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Biochemistry Test 1 2


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Amphiphilic / Amphipathic
Have one end that will dissolve in water and one end that wil disoolve in a nonpolar environment
Hydrogen Bonds
Hydrogen atom covalently bonded to one electronegative atom interacts with another electronegative atom
In SDS-PAGE, separation takes place on the bases of:
The sieving action of the gel, since all particles have approx the same charge/mass ratio, but dif. masses
A single water molecule can participate in up to __ H-bonds
The dominant interaction that drives a water-soluble protein to fold is:
The hydrophobic interaction
Which substance would be a suitable buffer at pH 5.0?
one with a pKa of ~ 5.0
Two amino acids frequently found in reverse turns are:
glycine and proline
In isoelectric focusing gel electrophoresis:
there is a pH gradient that parallels the electric fild gradient
True statements about water and/or H-bonds
1. H-atoms of water each bear a partial pos charge
2. H-bonds can form b/w diff parts of polypeptide chain
3. H-bonds are relatively weak compared to covalent bonds.
4. H-bonds in water contribute to its cohesiveness
Procedure necessary to test whether amino acid sequence of ibonuclease contains al the information needed for it to fold into its native, fucnal 3-d structure
1. FIRST: add 8M urea; RESULT: denatures, partially unfolds protein
2. FIRST: add reducing agent; RESULT: breaks S-S, RNase now completely unfolds
3. FIRST: dialyze away urea; RESULT: removal of denaturant, partial re-folding
4. FIRST: gendle air oxidation; RESULT: disulfides reform, final correct folding, yielding native form
Find the pH of 1L solution containing 0.52M sodium acetate and 0.48M acetic acid. The pKa of acetic acid is 4.76
pH = pKa + log[A-]/[HA]
= 4.76 + log 0.52/0.48
= 4.76 + 0.035
pH = 4.79
Find the pH of a solution after addition of 0.04 moles of HCl. Assume that HCl addition causes no change in volume.
HA <===> H+ + A-
[A-] = 0.52 - 0.04 = 0.48
[HA] = 0.48 + 0.04 = 0.52
pH = pKa + log0.48/0.52
= 4.76 - 0.035
pH = 4.73
Non-Covalent Interactions
1. H-bonds: b/w polar groups
2. Electrostatic (ionic): b/w charged groups
3. Hydrophobic: among non- polar groups in aq. solut'n
4. Van Der Waals
Delta G0
Standar Free Energy: Constant based on standard conditions --> additive for seuential reactions
Delta G
Actual Free energy change for eaction and function of concentration of products and reactant; fcn of temperature
Ionizable Protein Groups
1. terminal a-carboxyl group
2/3. aspartic & glutamic acid
4. Histidine
5. terminal a-amino group
6. Cysteine
7. Tyrosine
8. Tyrosine
9. Lysine
10. Arginine
pKa of terminal a-carboxyl group
pKa of Aspartic/Glutamic acid
pKa of Histidine
pKa of terminal a-amino group
pKa of Cysteine
pKa of Lysine
pKa of Tyrosine
pKa of Arginine
Gobular proteins
Gobular proteins are involved in the chemical functions of living things. The ones known as enzymes are organic catalyses that speed up the chemical reaction rates in cells. Other important gobular proteins are hemoglobin, antibodies, and some hormones.
Primary structure
There are four levels of structure in proteins. The simplest is level is called primary structure. It is determined by the kind, number and sequence of amino acids. The amino acids or peptides are joined to one another by strong covalent bonds. These bonds form between the nitrogen atom of the amino group and the oxygen atom of the carboxyl group. They are called peptide bonds.
Secondary structure
The second level of structure involves the coiling of primary chains into a helix or the formation of what are called pleated (beta) sheets Collagen is an example of a pleated sheet. Bonding between sulfhydryl groups of the amino acid cystine forms a strong covalent bond called a disulfide bond. In addition, ionic and weak hydrogen bonds help to stabilize the secondary structure.
Tertiary structure
The third level of protein structure has increased folding between alpha helix and non alpha helix regions. The structure is stabilized by disulfide, ionic, and hydrogen bonding. Gamma gobulin is an example of a gobular protein exhibiting tertiary structure. Keratin is a fibrous protein found in animal hair, claws and fingernails.
Quaternary structure
The final level of protein structure is composed of sub units of polypeptide chains with or without non amino acids. Like the two levels before them they are held together by disulfide, ionic and hydrogen bonding. Hemoglobin exhibits this structure. It is also an example of a conjugated protein because of the presence of a non amino acid group called the "heme group".
Denaturating of a Protein
The term denaturation is used to describe changes in a protein's shape. Such changes maybe temporary or permanent. In either case the breaking of the stabilizing bonds of a protein leads to its inactivation.

Any of the following conditions can bring about the denaturing of a protein:

* heat
* pH
* pressure
* chemicals
* heavy metals
Hydrophobic "Effect"
Other molecules exclude water molecules b/c of organization necessary to deal w/ H-bonds.
Hydrophobic "Effect" in Relation to DNA
Hydrophobic effect stabilizes stacking of base pairs in a linear piece of DNA;
Backbone (sugar) of DNA ar neg. charged, Electrostatic forces keep them apart and neutralized
Zwitterionic Form
Amino acids at neutral pH exist mostly as dipolar ions
-> amino is protonated (--NH3+)
-> carboxyl is de (--COO-)
Amino Acids in Acidic Solution
-> amino is prot'd (--NH3+)
-> carboxyl is prot'd (--COOH)
Amino Acids in Basic Solution
-> amino is deprotonated (--NH2)
-> carboxy is also de (--COO-)
Directionality of A.A. sequence
(N-term) (C-term)
Low molecular weight molecule such as glucose and glycerol
Epigenetic Factors
Associated w/ the genome by not represented in sequence of DNA
A unit of mass nearly = to that of a hydrogen atom
B-Pleated Sheet Stabilization & Bonds
* Stabilized by H-bonds b/w polypeptide strands
* Distance b/w AA = 3.5A
* Side chains of AAs point in opposite directions
Antiparallel B-Sheets
Adjacent B-sheets run in opposite direction
-> H-Bonds form b/w NH & CO groups; connect each AA to a single AA on an adjacent strand
Parellel B-Sheets
Adjacent B-sheets run in the same direction
-> H-bonds connect each AA on one strand to TWO different AA on another strand
* slightly less stable than antiparallel b/c of H-bond angle strain
pKa of carboxyl group
pKa of aspartic acid / glutamic acid
pKa of amino group
Phi angle
Torsion angle between a-carbon and nitrogen
Psi angle
Torsion angle between a-carbon and carboxyl carbon
D-Right // L-Left
* Refers to plane polarized light; uniform w/ respect to Z dimension; L vs D is determined by passing p.p. light through solution of molecule
Primary component of wool and hair.
* Consists of 2 right-handed a-helices intertwined to form a left handed helix: a-coiled coil
* Two helices are cross-linked by van der waals (if repeated residues are hydrophobic) & ionic (if repeated residues are hydrophilic) & disulfide (from cysteine)
* # of cross-links determine flexability
Collagen Helix
Rod-shaped; 3-helical polypeptide chain; GLY every 3 residues
* no H-bonds, instead stabilized by steric interactions/repulsion
Denatured Protein
Protein converted into a randomly coiled peptide w/o its normal activity
Sequence Specifies Conformation
The information needed to specify catalytically active structure of ribonuclease is contained in its amino acid sequence -> this structure is generally thermodynamically favored.
Chaperone Proteins
Proteins inside cells that prevent wrong folds under normal conditions
-> get energy from ATP
When will a protein conform to an a-helix?
"Default" Conformation
-> esp sequences including
When will a protein conform to a b-sheet?
* Residues w/ VAL, THR, ILE, because branching disrupts a-helix
* Also when side chains contain H-donors * acceptors that interact w/ main chain
When will there be a reverse turn?
Generally, when there's a proline -> ring structure is restrictive to a-helix or b-sheet.
Cooperative Transition
Proteins tend to be either all folded or all unfolded
-> protein placed in condition where some part is thermodynamically unstable: part of folded structure is disrupted -> interact'ns b/w it and remainder of protein are lost -> destabilizes entire protein
Leventhal's Paradox
Difference b/w calculated and actual folding time of a protein
-> Reveals that proteins do not fold by trying every conformation
Cumulative Selection of Protein Folding
* Partly correct intermediates are retained
-> correctness is not based on residues per se, but on total free energy
-> proteins are only marginally stable
Nucleation Condensation Model (Perscht) of Protein Folding
* A difuse folding nucleus is formed & consolidated through the transition state
-> local regions taht have significant structural preference, though not nec. stable on their own, will tend to adopt their favored structure
->interact: increase stability
* Viewed as a funnel: begins w/ highest free energy & many possible conformations; as free energy decreases, so do possible accessible conformations
Triggers phosphorylation of -OH of TYR
Matthews ('95)
Relationship b/w protein stability & protein function
* showed by residue substitution that increased stability reduces activity.
-> proteins fold to minimize free energy
-> organize themselves to recognize ligand
How is Stability of Protein Achieved?
Hydrophobic inside
Hydrophilic outside
Priority of Functional Groups
SH > OH > NH2 > COOH > CHO > CH2OH > C6H5 > CH3 > H
Anfinsen (1952)
First demonstrated that primary structure determines 3-D shape
* RNaseA (really hard to denature) & urea to denature & DTT to reduce disulfide bonds
-> showed RNaseA could complex w/RI
-> then used dialysis to renature -> regained activity
represents the fcnal expression of information, such as type, fcn, & interactions w/ protein that yield as a fcnal unit
Salting Out (precipitation)
Can be used to fractionate proteins since most are less soluble at high [salt]. This concentration (at which protein is no longer soluble) is diff for diff proteins
-> use [diff] depending on what you want to precipitate
Semipermeable membrane -> proteins too big to leave bag, while small molecules may leave
* useful to get rid of salt, although it won't sep diff proteins
Gel Filtration Chromatography
* gel (such as agarose) contains many beads, large molecules flow around them, small molecules get stuck in them
Ion Exchange Chromatography
CHARGE: (+) = SLOW, (-)=FAST
* gel contains neg charged beads (which contain carboxylate group); pos proteins will bind and take longer to run. (can also contain beads w/ (+) charge)
Affinity Chromatography
* separat'n based on a highly specific biologic interaction such
-> antigen and antibody
-> enzyme and substrate
-> receptor and ligand.
Proteins containing HIS tags are passed through a column of beads containing covalently attached, immobilized nickel (II) or other metal ions
-> HIS-tag binds tightly to immobilized metal ions, binding desired protein while other proteins flow though the column
* protein eluted by addition of imidazole or another molecule that displaces his
* similar to other column techniques but column materials are much finer
-> more interaction sites = greater resolving power
-> pressure added to obtain adeq. flow rate
Gel Electrophoresis
* run on polyacrylamide gel

f-depends on mas and shape and viscosity of medium)
E=electric field strength
Direction to run Gel Electrophoresis
(-) --> (+)
To separate based on mass only
1. SDS (anionic detergent): denatures by disrupting noncovalent interactions
2. mercaptoEtOH: reduces disulfide bonds
3. Anion of SDS binds to main chain
* Complex now has large net (-) charge -> runs based on mass
* separate w/electrophoresis
* visualize w/coomasie blue
* if radioactively labeled, arg (x-ray film over gel)

Why do we use a detergent?
Provides uniform charge to the protein (makes all neg.) by binding to peptide backbone stoichiometrically -> all proteins run towards (+), not back off the gel
Detergent used in SDS-PAGE
sodium dodecyl sulfate
Na+ -O-S-O-(CH2)-CH3
Names Helices
#residues/turn followed by # atoms in ring formed by H-bond
ex:a-helix=3.6 13
3 10 =tighter, narrower, longer
* Pretty rigid
* 4AA, often contain PRO & GLY
* small
* simply there to change direction
* bigger
* often flexible, sometimes not visible on x-ray
Induced Fit
often unstructured loop will acquire structure when interactions w/ another protein
Protein Domain
Series of discrete secondary structure in a singe peptide chain (~string of pearls)
-> often domains have specific/unique fcn or can fold independently
Isoelectric Focusing
* establish pH gradient & add voltage
* proteins stop moving at their isoelectric point (pH at which net charge is zero)
Overexpression System
Grow protein in non-native (foreign) organism
-> hRI is produced in E.coli
* Plasmid uses genetic elements from nature to control expression

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