1
Q
What is the central dogma?
A
describes the flow of genetic information (DNA->RNA->Protein)
2
Q
What are the two exceptions to the central dogma?
A
RNA viruses that use RNA as their genetic material and non-coding RNAs that do not produce proteins
3
Q
What did Francis Crick hypothesis about protein needs?
A
When a protein is needed a section of DNA is transcribed into RNA and is then read to produce a protein
4
Q
What is mRNA?
A
messenger rna
5
Q
What does mRNA do?
A
carries the instructions for building a specific protein to the ribosomes
6
Q
When is mRNA synthesized?
A
during transcription where RNA pol reads DNA and repairs RNA bases
7
Q
What is rRNA?
A
ribosomalnRNA
8
Q
What does ribosomal RNA do?
A
acts as components o the ribosome which is required fro translation (most abundant RNA in the cell)
9
Q
What is tRNA?
A
transfer RNA
10
Q
What does tRNA do?
A
transfers individual amino acids from the cytoplasm to their appropriate location in the growing polypeptide chain during protein synthesis
11
Q
What are the parts of a nucleic acid?
A
a nitrogenous base, a pentose sugar, and at least one phosphate group
12
Q
What are purines?
A
Two ringed structures (A and G)
13
Q
What are pyrimidines?
A
Single ring structures ( C U T)
14
Q
What are the 3 fundamental differences between DNA and RNA?
A
DNA is ds
DNA contains deoxyribose
DNA contains thymine rather than uracil
15
Q
What bonds link nucleotide monomers?
A
phosphodiester bonds between the 5’ phosphate an d the 3’ hydroxyl
16
Q
What is the amino group?
A
a group with 1-3 hydrogens depending on peptide bond participation and pH
17
Q
What is the carboxyl group?
A
negative at physiological pH, and a hydroxyl is lost in participation in a peptide bond
18
Q
What is the R group?
A
each amino aid has a unique side chain bonded to the alpha carbon
19
Q
What is the directionality in polypeptides?
A
N to C terminus
20
Q
What is electronegativity?
A
A measure of the likelihood of formation of a covalent or ionic bond?
21
Q
What does the difference between electronegativity values represent?
A
The character of the bond
22
Q
What are salt bridges?
A
interaction between oppositely charged amino acid side chains which involves hydrogen bonding and electrostatic interactions
23
Q
What are weak bonds?
A
Bonds involving.greater distances between atoms, easily broken, and are transient
24
Q
What are the three types of weak interactions?
A
van der Waals force, hydrophobic interaction, and hydrogen bonds
25
What are van Der Waals force?
non-specific contacts between atoms where proximity induces weak fluctuating charges. interaction becomes repulsive if atoms becomes too close
26
How must molecules fit to effectively use van Der Waals forces?
Exactly, where the distance between any two atoms must not be different than the sum of their van Der Waals radii
27
What is the hydrophobic effect?
The exclusion on non polar groups from polar ones, resulting in aggregation
28
Why is hydrophobicity energetically unfavourable?
It decreases entropy
29
What is pi stacking?
Attractive non covalent interactions between aromatic rings (such as between adjacent DNA bases)
30
When do pi interactions occur?
When aromatic or conjugated systems, stack with their p bonding systems layered
31
What is the directional preference of a hydrogen bond?
a straight line
32
How are complementary nucleic acid strands held together?
By hydrogen bonds
33
What is the purpose of the major and minor grooves?
hydrogen bonding with proteins
34
What are proteins composed of?
amino acids
35
How are amino acids categorized?
by their chemical properties determined by their side chain's polarity and chagre
36
What are the five classifications of amino acids?
non-polar aliphatic, non-polar aromatic, polar uncharged, negatively charged, and positively charged
37
What are aliphatic side chains?
Side chains that are conly composed of hydrocarbons, which are non-polar and quite hydrophobic
38
Where are non polar aliphatic amino acids usually found within a protein?
usually in the interior of the protein, which is a major driving force behind protein folding
39
Describe glycine?
Hydrogen side chain. very small impact on hydrophobic effects
40
Describe alanine, valine, leaucine, and isoleucine
Side chains made up of simple hydrocarbons. Leucine and isoleucine are regioisomers
41
Describe methionine
The side chain contains a thioether group (C-S-C)
42
Describe proline?
proline is the only amino acid whose side chain con nests to the amino group, creating a 5=membered ring, causing rigidity and limiting conformations
43
What are aromatic side chains?
side chains with aromatic substituents. phenylalanine is the most hydrophobic, while tyrosine and tryptophan have polar substituents. all participate in hydrophobic interaction
44
What property of proteins is determined by aromatics?
absorption of ultraviolet light at 280nm
45
What are polar uncharged side chains?
interact extensively with water or with other atoms in other side chains through h bonds
46
Describe Cysteine
contains a sulfhydryl ground (R-S-H) which may oxidize to form a disulphides bond when in proximity with other cyteines. This acts as a molecular cross-brace, enhancing protein stability. important for catalysis as the deprotonated -SH is a good nucleophile
47
What did Matthew demonstrate in 1989 at the University of Oregon?
The structural importance of the disulphide bonds by artificially adding sysgtein residues to the enzyme T4 lysozyme
48
What procedure did Matthew use?
3 pairs of Cys residues were engineered into a T4 lysozyme creating a mutant with 3 disulhide bonds
49
What were the results of Matthew's experiment?
additional disulphides bonds stabilize the protein structure relative to wild type lysozyme using temperature at which the protein loses function as a metric
50
Describe asparagine and glutamine?
contains amide groups that can act as donor or acceptors of electrons, along with C=O groups that can participate in H-bonding and dipole dipole interaction
51
escribe Serine and Threonine
OH group in their side chains, capable of forming hydrogen bonds. These may also be phosphorylated (along with OH on tyrosine) by kinases during protein modification
52
What are negative side chains?
side chains with a carboxyl group that are deprotonated at physiological pH. They are acidic. aspartic and glutamic acid
53
What are positive side chains?
They are basic. lysine, arginine and histidine
54
What does lysine contain?
amino group
55
What does arginine contain?
A guanidium group
56
What does histidine contain?
An imidazole group
57
Where are charged amino acids located?
exterior of protein due to hydrophilicity
58
Why are charged amino acids important for enzyme function?
They are able to interact with other charged molecules and are commonly located in the active site
59
What are zwitterrions/
Molecules that are neutral but with both a positive and a negative charge
60
How are amino acids zwitterions?
They contain a positive amino group and a negative carboxyl group at neutral pH, meaning they can both accept and donate protons
61
How is the ionization state on the amino, carboxyl, and R groups determined?
by the pH of their surroundings
62
How can the acid base behaviour of a peptide be determined?
By its free alpha amino and alpha carboxyl groups combined with the nature and number of its ionizable R groups
63
What are the pKa values of aspartate, glutamate, arginine, and lysine?
low for the negative amino acids and high for the positive amino acids
64
What is pH?
power of hydrogen. A measure of the hydrogen ion concentration
65
What is the pH of normal human blood and tissues?
7.4
66
How much can the pH of human blood or tissues fluctuate before it is life threatening?
+/- 0.2 as nucleic acids and proteins depend on regulated pH
67
Why is pH important?
It influences the structure and function of many enzymes in living systems.
68
What pH does pepsin work best at and why?
pH of 2 as the carboxylic acid at the enzymes active site must be protonated
69
How do our bodies regulate the pH of our bloodstream?
Via a buffer system
70
What is an example of the buffer system in the body?
lungs and blood. CO2 is produced in our bodies which combines with water to make carbonic acid which partially dissociates into hydrogen and bicarbonate. When the blood reaches the lungs the carbon dioxide comes out of the solution and is expelled
71
Describe the ionization of glycine?
exists as a zwitterion at neutral pH, turns into a diprotic acid at low pH, at high pH. it is fully deprotonated
72
What happens when pH=pKa?
50% of the molecules in concentration have a deprotonated carboxyl and 50% do no
73
What happens when pH>pKa of the carboxyl group?
a molecule with a non-ionizable R group will be predominantly zwitterionic
74
What occurs when pH=pKa of the amino group?
50% are zwitterions and 50% are fully deprotonated
75
What is the pKa range of a weak acid?
-2 to 12 in water
76
What is the pKa range of a strong acid?
<-2
77
What is the Henderson-Hasselbalch Equation?
Defines the relationship between pH and the ration of A to HA. pH=pKa+log [A]/[Ha]. pH>pKa, [A] to [HA]>1, and vice versa
78
What happens when pH=pKA?
50:50 mixture of A and HA
79
How can the pKa of an acid be measured?
by performing a titration
80
How is a titration performed?
Bye addition of a base in increments which are recorded, plotted against pH. The curve will yield the pKa and the midway point
81
What is the pKa of the carboxyl group?
about 2
82
What is the pKa of the amino group?
9.5
83
What is the pKa of an R group called?
pKr
84
What are type I groups?
groups that are neutral in acidic form and lose their proton when pH is greater than pKa. Carboxyl group for example
85
What are type II groups?
positively charged in acidic form and lose their proton when pH is greater than pKa. examples include the amino groups, histidine side chain, and the arginine side chain
86
What is the isoelectric point?
pI is the pH at which an amino acid or polypeptide is ionized and has no net charge
87
How is pI calculated?
by averaging the pKa values for the ionization states thet straddle the electrically neutral species (pI=pKa1+pKa2/ 2)
88
What type of reaction is peptide bond formation?
a dehydration reaction that involves the loss of a water molecule (OH from C terminus and H from N terminus)
89
What is primary protein structure?
the linear sequence of amino acids read N to C
90
What are the constraints on the peptide backbone?
There is little free rotation about the C-N bond between two alpha carbons. This is due to resonance, the planar nature of the 4 atoms involved in the peptide bond, and the short C-N bond length, which approaches the length of a C=N bond.
91
Is there any rotation in the peptide backbone?
yes. between the N-C(a) bond and the C(a)-C bonds. The andes are constratined however.
92
What are torsion angles?
the rotational angels in peptides. phi for th N-C bond and Psi for the C-C bonds
93
Why are Phi and Psi angels limited?
Because of steric clash by R groups and neighbouring atoms
94
What is a rakmachandran plot?
a plot of the allowed angles of phi and psi for each amino acid. Similar looking for most amino acids
95
What are the two exceptions to most rakmachandran plots looking alike?
glycine due to the side chain being just hydrogen and proline due its cyclic structure being greatly restrained
96
What are favourable angles of rotation?
conformation that involve little or no interference between atoms based on known van Der Waals radii and bond angels. These are represented by shaded areas on rakmachandran plots with darker color representing easily allowed angles
97
What are secondary structures of proteins?
Regularly repeating elements within a protein in which h bonds form between polar atoms in the backbone chain. Mainly alpha helices (10-15 residues) and B sheets composed of individual strands of 3-10 residues
98
How much of a protein is typically alpha helices or B sheets?
1/3 respectively. There are plenty of exceptions however
99
What is the alpha helix?
The most stable arrangement of polypeptide backbone. It is a right handed helix with 3.6 was per turn. A full turn is 5.4 A long with the R groups protruding outward
100
What is the pattern of H bonding in alpha helices?
Hydrogen on the amide nitrogen forms a hydrogen bond with the carbonyl oxygen of the fourth residue towards the N terminus
101
What is the B sheet?
second most stable structure. H bonds between the backbone amide and carbonyl groups. h bonding between two different strand. R groups alternate resulting in a bzigzag pattern involving 4-6 strands
102
What kinds of amino acids can B sheets readily accommodate?
aromatic residues and proline which is unflavoured in alpha helix.
103
What can B sheets act as a boundary between greasy and watery environments?
Because alternating R groups are on opposite sides of the sheet, and non-polar alternating with polar can act as this boundary
104
How can rakmachandran plots identify the secondary structures in a protein?
As each secondary structure has a characteristic range that of psi and phi values that is allowed
105
What are reverse turns?
turns that reverse the direction and fold of a protein to allow tertiary structures. Sometimes large and irregular but usually small and precise
106
What are B turns?
small and precise reverse turns
107
How many residues is needed for a B turn to make a complete turn ?
4. The carbonyl of the first H bonds with the amide hydrogen of the 4th. No tier-residure h bonds in 2 and 3, so they h-bond to water on the outside of the protein. Usually a proline at 2 and a glycine at 3.
108
Why are glycine and proline found at 2 and 3 in B turns?
glycine is a single hydrogen which allows it to accomodate many conformations. Proline is highly restricted but the amino nitrogen of Pro can readily assume a cis configuration. Especially suitable for tight turns
109
What is the gamma turn?
A much less common three amino acid turn where the backbone carbonyl and amide groups of the first and third amino acid residues for a h bind. The middle is no involved in inter residue h bond
110
What is the tertiary structure of proteins?
Packing or folding 3D
111
What are globular proteins?
water soluble and spherical proteins. Ex. hemoglobin
112
What are fibrous proteins>
proteins with elongated shapes and play a structural role in the body. Ex. collagen, a supportive connective tissue network, and keratin, a protective protein
113
When do proteins fold?
spontaneously under physiological conditions as they are biosynthesized
114
What is the quaternary structure of proteins?
defined by the connections between two or more polypeptide side chains.
115
Why is the formation of multi subunit complexes favourable with reference to folding?
If a domain misfiles, the entire protein would be nonfunctional and cellular energy would be wasted. IF one subunit misfiles, it will not be included in the oligomer and only the investments of one domain was wasted. If one subunit denatures or becomes inactive, it can also be replcaed
116
What is an oligomer/multimer?
a protein composed of multiple polypeptides
117
What is a subunit/protomer?
An individual polypeptide
118
What is a homooligomer?
an oligomer composed of identical subunits
119
What are domains?
separate folding units often formed by single proteins larger than 150-200 residues
120
Where are the secondary structures that comprise a domain often found in the primary sequence?
next to each other although this is not always the case
121
Can domains perform different or the same functions?
Yes both
122
Can domains interact?
yes
123
How are the boundaries between domains usually assessed?
By examining the proteins structure or through proteolyssi
124
Once a protein si folded is it fixed?
No the structure is constantly in flux with the structure vibrating rapidly. This is essential for protein function
125
What is the native state of the protein?
selected for at amino acid level so that it is the lowest energy state in that form. Promotes the correct folding of proteins so that they serve the same function in future generations
126
How does regular folding occur?
intermediates can form which are low in energy, however chaperones and chaperonins exist to move it to the native state
127
What are misfolds?
states that are lower in energy than the native state in which the protein may get stuck leading to essentially irreversible aggregations or amyloid fibrils which cause neurological disease
128
Why is predicting protein folding not possible yet?
Due to the complexity of the protein folding code and the small differences in free energy between folded and unfolded states
129
What is the naming convention used with promoters
a b y for different subunits that make up a protomer
130
What is protein purification?
The process of isolating and purifying a protein of interest
131
What are the main considerations in protein purification?
Fact: any one protein is usually present as a very small epicene takeoff the total protein
Goal: separate the protein from others\
Preparation: The more you know about the proteins properties, the better strategy you can make
Method consideration: biological source, extraction conditions, number of steps, and desired use
132
What are the four main steps in the purification of proteins?
cell lysis, cetrifugation, fractionation, and protein detection
133
What are the four most common methods of cell lysis?
detergent, shear force, low ionic salt, changes in pressure
134
How do detergents work?
Compromise the integrity of the cell membrane
135
How does shear force work?
high frequency sonic waves to agitate and break the membrane. Rapidly shaking the sample also works
136
How does low ionic salt work?
Treatment of cell cultures with low ionic salt concentrations causes cells to osmotically absorb water and pop
137
How do changes in pressure work?
high amounts of pressure can cause breakage
138
How does centrifugation work?
By taking advantage of the sedimentation principle, where radial acceleration causes denser particles to sediment at the bottom (the pellet) and low density rises to the top (the supernatant)
139
How does differential centrifugation work?
repeated centrifugation cycles at different rotational speeds to separate increasingly less dense material from denser particles
140
What is the crude extract?
soluble proteins following the removal of cell wall debris and insoluble proteins. This can then be fractionated by additional methods to obtain the protein of interest
141
What is column chromatography?
separation of a mixture of compounds by dissolution in a fluid known as the mobile phase which is then passed through a structure containing another material known as the stationary phase. Separation occurs due to the stationary phase causing different components to travel at different speeds
142
What are the steps in column chromatography?
1. A protein mixture is applied to a column containing a resin or metric that interacts differently with the various proteins
2. buffer is passed through the columns to wash away any proteins that do not bind to the matrix
3. another buffer that causes bound protein to dissociate from the Matrix is applied, and the proteins are carried out in the buffer flow through a process called elution. This occurs at different times depending on the interaction with the resin
4. The eluted proteins are collected in a fraction collector which gradually moves test tubes under the column, thus keeping the protein that elute at different times separate from one another
143
What are the three main types of column chromatography?
ion-exclusion chromatography,
size exclusion chromatography
affinity chromotography
distinguished by the types of restn in the column
144
What is ion exchange chromatography?
separating ions by charge
145
What does the resin of ion exchange chromatography contain?
Either an anion exchange resin which is positively charged or a cation exchange resin which is (you guessed it) negatively charged
146
How are proteins usually eluted in ion exchange chromatography?
Proteins are usually eluted from the column with an increasing concentration of salt solution and their release depends on the nature of the charged amino acid residues on their surface (ie with a cation exchange resin, the more negative proteins will elute earlier and faster
147
How are proteins bound in ion exchange chromatography?
when the pH of the solution surrounding the protein is lower than the pI then the protein has increased cationic character and will have a net positive charge
148
How are proteins eluted in ion-exchange?
by adding counter-ions to the bound proteins on the column, these compete for the ionic interactions the proteins make
149
What is size exclusion chromatography?
The column matrix contains beads that have pores of specific size which allows proteins smaller than the pore to enter. Therefore the proteins that enter the pores will take longer to migrate through the column while larger proteins will move around the pores elute earlier
150
What is affinity chromatography?
takes advantage of the fact that many proteins specifically bind other molecules or ligands as part of their function. This can be used to construct a column containing the ligand covalently attached to a matric
151
How can proteins be made to undergo affinity based purification?
By addition of short chain amino acid sequences to the beginning or end of the target protein by molecular cloning method
152
What is a Glutathione-S-transferase tag?
GST is a protein that rapidly folds into a stable and highly soluble protein upon translation. Therefore inclusion of the GSt tag often promotes greater expression and solubility of recombinant proteins. GST also binds to its substrate, glutathione which is a tripeptide (E-C-G). Glutathione immobilized to a resin can allow fro enzyme substrate binding
153
What is SDS-PAGE?
sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. Uses an electrical current to separate proteins according to their size.
154
What is the acrylamide content of polyacrylamide gel?
6-10%. high percent gels resolve small proteins better
155
What is SDS?
A negatively charged detergent that binds proteins and denatures them, giving all proteins a similar shape. Because amount of SDS binding to a protein is usually related to protein size, SDS also gives all proteins a similar change to mass ratio
156
What is the first step of SDS PAGE?
Treated samples are loaded into the wells at the top of the gel and an electrical field is applied to the gel which pulls the charged protein through the matrix. SDS is negative so the proteins will move towards the positive cathode at the bottom o the few
157
What is the second step of SDS PAGE
The proteins migrate at different rates by relative molar mass. Small ones migrate faster
158
What is step 3 of SDS page?
gel is removed from between two pieces of glass which hold and and it is soaked in an acidic buffed to fix the proteins from diffusing out of the gel. The gel is then treated with a dye that selectively binds to proteins
159
BCHM 218
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