Biochemistry Test 1

Front 1

Amphiphilic / Amphipathic

Back 1

Have one end that will dissolve in water and one end that wil disoolve in a nonpolar environment

Front 2

Hydrogen Bonds

Back 2

Hydrogen atom covalently bonded to one electronegative atom interacts with another electronegative atom

Front 3

In SDS-PAGE, separation takes place on the bases of:

Back 3

The sieving action of the gel, since all particles have approx the same charge/mass ratio, but dif. masses

Front 4

A single water molecule can participate in up to __ H-bonds

Back 4

4

Front 5

The dominant interaction that drives a water-soluble protein to fold is:

Back 5

The hydrophobic interaction

Front 6

Which substance would be a suitable buffer at pH 5.0?

Back 6

one with a pKa of ~ 5.0

Front 7

Two amino acids frequently found in reverse turns are:

Back 7

glycine and proline

Front 8

In isoelectric focusing gel electrophoresis:

Back 8

there is a pH gradient that parallels the electric fild gradient

Front 9

True statements about water and/or H-bonds

Back 9

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

Front 10

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

Back 10

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

Front 11

Find the pH of 1L solution containing 0.52M sodium acetate and 0.48M acetic acid. The pKa of acetic acid is 4.76

Back 11

pH = pKa + log[A-]/[HA]
= 4.76 + log 0.52/0.48
= 4.76 + 0.035
pH = 4.79

Front 12

Find the pH of a solution after addition of 0.04 moles of HCl. Assume that HCl addition causes no change in volume.

Back 12

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

Front 13

Non-Covalent Interactions

Back 13

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

Front 14

Delta G0

Back 14

Standar Free Energy: Constant based on standard conditions --> additive for seuential reactions

Front 15

Delta G

Back 15

Actual Free energy change for eaction and function of concentration of products and reactant; fcn of temperature

Front 16

Ionizable Protein Groups

Back 16

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

Front 17

pKa of terminal a-carboxyl group

Back 17

3.1

Front 18

pKa of Aspartic/Glutamic acid

Back 18

4.1

Front 19

pKa of Histidine

Back 19

6.0

Front 20

pKa of terminal a-amino group

Back 20

8.0

Front 21

pKa of Cysteine

Back 21

8.3

Front 22

pKa of Lysine

Back 22

10.8

Front 23

pKa of Tyrosine

Back 23

10.9

Front 24

pKa of Arginine

Back 24

12.5

Front 25

Gobular proteins

Back 25

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.

Front 26

Primary structure

Back 26

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.

Front 27

Secondary structure

Back 27

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.

Front 28

Tertiary structure

Back 28

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.

Front 29

Quaternary structure

Back 29

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".

Front 30

Denaturating of a Protein

Back 30

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

Front 31

Hydrophobic "Effect"

Back 31

Other molecules exclude water molecules b/c of organization necessary to deal w/ H-bonds.

Front 32

Hydrophobic "Effect" in Relation to DNA

Back 32

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

Front 33

Zwitterionic Form

Back 33

IN NEUTRAL pH!
Amino acids at neutral pH exist mostly as dipolar ions
-> amino is protonated (--NH3+)
-> carboxyl is de (--COO-)

Front 34

Amino Acids in Acidic Solution

Back 34

-> amino is prot'd (--NH3+)
-> carboxyl is prot'd (--COOH)

Front 35

Amino Acids in Basic Solution

Back 35

-> amino is deprotonated (--NH2)
-> carboxy is also de (--COO-)

Front 36

Directionality of A.A. sequence

Back 36

AMINO -> -> CARBOXYL
(N-term) (C-term)

Front 37

Metabolite

Back 37

Low molecular weight molecule such as glucose and glycerol

Front 38

Epigenetic Factors

Back 38

Associated w/ the genome by not represented in sequence of DNA

Front 39

Dalton

Back 39

A unit of mass nearly = to that of a hydrogen atom

Front 40

B-Pleated Sheet Stabilization & Bonds

Back 40

* Stabilized by H-bonds b/w polypeptide strands
* Distance b/w AA = 3.5A
* Side chains of AAs point in opposite directions

Front 41

Antiparallel B-Sheets

Back 41

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

Front 42

Parellel B-Sheets

Back 42

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

Front 43

pKa of carboxyl group

Back 43

3.1

Front 44

pKa of aspartic acid / glutamic acid

Back 44

4.1

Front 45

pKa of histidine

Back 45

6.0

Front 46

pKa of amino group

Back 46

8.0

Front 47

pKa of cysteine

Back 47

8.3

Front 48

pKa of lysine

Back 48

10.8

Front 49

pKa of tyrosine

Back 49

10.9

Front 50

pKa of arginine

Back 50

12.5

Front 51

Phi angle

Back 51

Torsion angle between a-carbon and nitrogen

Front 52

Psi angle

Back 52

Torsion angle between a-carbon and carboxyl carbon

Front 53

(D)extroratotory
(L)evoratatory

Back 53

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

Front 54

a-Keratin

Back 54

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

Front 55

Collagen Helix

Back 55

Rod-shaped; 3-helical polypeptide chain; GLY every 3 residues
* no H-bonds, instead stabilized by steric interactions/repulsion

Front 56

Denatured Protein

Back 56

Protein converted into a randomly coiled peptide w/o its normal activity

Front 57

Sequence Specifies Conformation

Back 57

The information needed to specify catalytically active structure of ribonuclease is contained in its amino acid sequence -> this structure is generally thermodynamically favored.

Front 58

Chaperone Proteins

Back 58

Proteins inside cells that prevent wrong folds under normal conditions
-> get energy from ATP

Front 59

When will a protein conform to an a-helix?

Back 59

"Default" Conformation
-> esp sequences including
* ALA
* GLU
* LEU

Front 60

When will a protein conform to a b-sheet?

Back 60

* Residues w/ VAL, THR, ILE, because branching disrupts a-helix
* Also when side chains contain H-donors * acceptors that interact w/ main chain

Front 61

When will there be a reverse turn?

Back 61

Generally, when there's a proline -> ring structure is restrictive to a-helix or b-sheet.

Front 62

Cooperative Transition

Back 62

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

Front 63

Leventhal's Paradox

Back 63

Difference b/w calculated and actual folding time of a protein
-> Reveals that proteins do not fold by trying every conformation

Front 64

Cumulative Selection of Protein Folding

Back 64

* Partly correct intermediates are retained
-> correctness is not based on residues per se, but on total free energy
-> proteins are only marginally stable

Front 65

Nucleation Condensation Model (Perscht) of Protein Folding

Back 65

* 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

Front 66

Insulin

Back 66

Triggers phosphorylation of -OH of TYR

Front 67

Matthews ('95)

Back 67

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

Front 68

How is Stability of Protein Achieved?

Back 68

Hydrophobic inside
Hydrophilic outside

Front 69

Priority of Functional Groups

Back 69

SH > OH > NH2 > COOH > CHO > CH2OH > C6H5 > CH3 > H

Front 70

Anfinsen (1952)

Back 70

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

Front 71

Proteome

Back 71

represents the fcnal expression of information, such as type, fcn, & interactions w/ protein that yield as a fcnal unit

Front 72

Salting Out (precipitation)

Back 72

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

Front 73

Dialysis

Back 73

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

Front 74

Gel Filtration Chromatography

Back 74

SIZE: LARGE=FAST, SMALL=SLOW
* gel (such as agarose) contains many beads, large molecules flow around them, small molecules get stuck in them

Front 75

Ion Exchange Chromatography

Back 75

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)

Front 76

Affinity Chromatography

Back 76

FUNCTIONAL GROUPS
* separat'n based on a highly specific biologic interaction such
-> antigen and antibody
-> enzyme and substrate
-> receptor and ligand.

Front 77

HIS-tags

Back 77

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

Front 78

HPLC

Back 78

* similar to other column techniques but column materials are much finer
-> more interaction sites = greater resolving power
-> pressure added to obtain adeq. flow rate

Front 79

Gel Electrophoresis

Back 79

NET CHARGE AND SIZE
* run on polyacrylamide gel

velocity=Ez/f
f-depends on mas and shape and viscosity of medium)
z=charge
E=electric field strength

Front 80

Direction to run Gel Electrophoresis

Back 80

RUN TO RED
(-) --> (+)

Front 81

To separate based on mass only

Back 81

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)

Front 82

Acrylamide

Back 82

Acrylamide

Front 83

Why do we use a detergent?

Back 83

Provides uniform charge to the protein (makes all neg.) by binding to peptide backbone stoichiometrically -> all proteins run towards (+), not back off the gel

Front 84

Detergent used in SDS-PAGE

Back 84

sodium dodecyl sulfate
O
"
Na+ -O-S-O-(CH2)-CH3
"
O

Front 85

Names Helices

Back 85

#residues/turn followed by # atoms in ring formed by H-bond
ex:a-helix=3.6 13
3 10 =tighter, narrower, longer

Front 86

Turns

Back 86

* Pretty rigid
* 4AA, often contain PRO & GLY
* small
* simply there to change direction

Front 87

Loops

Back 87

* bigger
* often flexible, sometimes not visible on x-ray

Front 88

Induced Fit

Back 88

often unstructured loop will acquire structure when interactions w/ another protein

Front 89

Protein Domain

Back 89

Series of discrete secondary structure in a singe peptide chain (~string of pearls)
-> often domains have specific/unique fcn or can fold independently

Front 90

Isoelectric Focusing

Back 90

CONTENT OF ACIDIC & BASIC RESIDUES
* establish pH gradient & add voltage
* proteins stop moving at their isoelectric point (pH at which net charge is zero)

Front 91

Overexpression System

Back 91

Grow protein in non-native (foreign) organism
-> hRI is produced in E.coli
* Plasmid uses genetic elements from nature to control expression