1
Q
How are amino acids typically arranged in proteins
A
- Nonpolar are found on inside for foldimg
- Polar water loving on the outside
- Negative charged are the acids which contain a C group that is negatively charged at pH
- Polar positive carry a charge a pH
- Nonpolar are aromatic r groups
*
2
Q
What are some properties of nonpolar amino acid aromatics
A
- Absorbs UV light of 280nm
- Bigger the aromatic the bigger the wavelength it can absorb
3
Q
What is the ionization of amino acids
A
- Exist as zwitterions - carry localized charges at pH
- Can accept or DOnate protons form amino they donate and accept from carboxyl group
- Not all amino acids are ionizable
- At neutral pH they are charged asn can act as a weak base or a weak acid
4
Q
What is H, OH and PH
A
- pH = -log(H+)
- very acidic means most H ions
- Basic means very little H protons
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5
Q
How is the strength of acids and bases determined
A
- Ka and pKa
- Ka is dissassociation constant, larger the Ka the easier it will disassociate
- pKa = -logKa
- Measure of tendency to gain or lose a proton
- Lower pJa the stronger the acid and the likihood of giving up protons and higehr teh easier it will accept a proton
- Ka = [A-][H3O+]/[HA]
6
Q
Describe how pH and amino acids function
A
- active site has glutamic acid that carries negative charge
- Allows approproate charge to bind
- Pepsin works best at Ph of 2, This keeps C acid group on the amino acid allowsing it to break chemical bonds outside of teh stonmach where the pH is larger and will not be functional
7
Q
WHat is the henderson hasselbalch equation and what does it do
A
- When pH is greater than pKa
- Ratio of basic to acuidic solutioons is greater than 1
- when it is less than pKa it is less than 1
- as pH increases there is less of the acidic form
8
Q
What are the type I and Type II amino aicds
A
Type i
* Neutral in acidic form
* Carry NEGATIVE CHARGED WHEN PH > PKA AND NEUTRAL WHEN PH < PKA
* Carboxyl group C terminal
* glutamate
* aspartate
* Tyrosine
* Cysteine
Type II
* Positively chagred in acific form
* Neutral when pH > Pka and positivly charged when pH < pKa
* Lysine
* histidine
* Arginine
* Amino group on all amino acids
9
Q
Lysine has pKr of 10.53 what its prediminant R group at charge pH 10
A
pH = pKa + log[A-]/[HA]
Since pH 10, there will be a deprotenation event
10 - 10.53 = log[NH2]/[NH3]
0.3 = [NH2/[NH3] = 3.33x more NH3 than NH2
10
Q
How do you find the net charge of a polypeptide
A
- Find what all the amino acids are
- Identify the ionizable grpups such as N terminals, C terminals and any R groups
- Classify them by type I and Type II and identify their behaviour
- Now add up all the charges
11
Q
What is the isoelectric point
A
- Pl = Pka1 + pka2/2
- when amino aicd is ionized yet electrically neutral
- take the pKa of the Carboxyl group and the amino acid
*
12
Q
How do you find the isoelectric point of an amino acid
A
- Idnetify ionible groups
- Terminals C and N groups are no longer ionizble in a peptide bond because water is lost
- Determine the charges of the ionible groups at different pH using the Type I and type II
- Calculate the pl with 2 values directly above abd below the pH where the net charge is 0
13
Q
What are salt bridges
A
- Involve 2 interactions
- H and eletcrostatic
- Happens between eletcrically charged amino acidic and basic groups
14
Q
What is said to be about the levels of protein striuctures
A
- They are nested - Higher levels are formed entirely of lower levels
15
Q
What is a primary structure of amino acids
A
- Linear sequence of amino acids
- read from N to C
- peptide bonds do not allow freedome of rotation from resonance
- Peptide bond shorter than regylar bond andb hevaes like a double bond
- Phi bond N-Calpha bond
- Psi for Calpha - C bond
- Both of these are free roration. In polypeptide they are constrained from the bulk of side chains
- Called steric clas between R groups and side chains and that is whats the ramachandron plot does
16
Q
What are key features of the ramachandron plot
A
- Top left has anti nad parallel beta sheets and right twisted ones
- Bottom left is right handed alpha helix
- Top righjt is lefr handed
- Exceptions are glycine which has very small R group so has many rotations and proline which has a huge R group which prevents rotation
17
Q
What are basic properties of secondary structures
A
- Regular repeating units and patters
- proteins are usually 1/3 alpha helix and 1/3 beta sheet
- Made of a lot of turns and sides
- They can be identifiers that require a specific turns to happen
18
Q
What is an alpha helix
A
- 3.6aa per turn
- 5.4 angstroms is 1 full turn
- H bonds that stabilize this reaction are slightly offset in the helix but they are generally parallel
19
Q
What is a beta sheet
A
- Second most stable form the alpha helix
- Unlike the alpha helix cannot without multiple strands
- They arwe pleated from the zigzag pattern to prevent interatcions with R groups of Amino acids
- Anti parallel is the most conformation
- There is also mix of parralel and anti parralalel
- then parralel which is the least common
- B sheets accommate large hydrophic aromatic amino acids and proline which is called a helix breaker
- alternating R goups allow polar and nonpolar to form a boundary between more water and greasy environment
20
Q
What are beta turns
A
- 1/3 of proteins and reverse the structure of the patterns
- uses 4 residues, first carboonyl H bonds with the 4th residue, the second residue is a proline becasuse it can go to a cis position and 4th is a glycine because it has the biggest free rotation
21
Q
What are the 3 type of teritiary structures
A
- Defined by folding of secondary structures
- Globular - water soluble and spherical
- Fibrous - elongated and stronger in shape
22
Q
WHat are quatrenary structures
A
- Connections of multiple polypeptides that create full proteins
- Monomer
- DImer
- Trimer
- Oligonomer
- multimer for respective dubunits which are prorteins
23
Q
What is the difference between a domain and a subunit
A
- Subinit is a single polyppetide within a protein
- Domain is a section of a polyppetide that has a discreet function and independent folding of the polypepitde
- protein 150-200 aa or have 2 or more domains may habe the same function or different function
- secondary structures that comprise domains are typucally adjacent to 1 nother by primary sequence
24
Q
How are proteins folded
A
- Polypeptides fold to adopt a particular conformation
- domaisn exist to minimize risk of misfolding. If a multi subuinit compostion prevents the entire protein from being inoperable. A single subunit can be replaced and used
- is sponteanous reaction becausei t lessens the energy state
- as a orotein fold they become lower energt
- they are foklding intermediates that are relativekt stabkle
- The protein chaperones will break them into their lower energy states
- Some folded proteins are higehr energy than their native state and aggregations that are irreversible neurotoxic
DIAGRAM - UNfolded proteins are highest energy
- Folding intemredates and nagtive states and partially folded are a bit lower energy
- Amorpheus aggregates are very low energy
- Oligonomers are the same energy as the iuntermediates and the amyoloid fibrils are very low energy the lowest
25
What are the general steps of protein purification
* Cell lysing - protein extraction and breaking apart the cell
* Centrifugation - seperating density of proteins
* fractionation - seperating the types of proteins
* Protein detection
26
Step 1 Cell lysis
* Breaking the cell to get whats needed
* Detergents comprise membrane integreity
* shear force - breaking the cell membrane physically by shaking
* Low ionic salt - causes high volumes of fluid to enter the cell froj osmosis and causes the cell to pop
* Chamnhes in pressure causes a rupture in the cell
27
Step 2 Centrifugation
* Radial rapid rotation causes high density particules to end up at the bottom and sediment loiw desnsity at the top
* Overtime the the speed wil be increases
* The crude extract is soluble proteins
28
Step 3 Fractionation
**Colour chromatograohy**
* Dissolve the miuxture in fluid alled the mobile phase has the protein of interest
* Applied in the colour nad moves through it which has a matrix that has affinity to the protein of interest
* Causes the proteins to travel at different speeds it is then eluted
**Ion exchange chromatography **
* Column contains a positive charge that binds to negative anion and vice versa
* Cation exchange binds to cations and so the columb must be negative
* In this case most positively charged proteins will exist last
* WHen the pH of solutoon is less than the Pl the protein will jhave increases cationic character
* when it is greater it will have anionic character
**Size exclusion chromatography **
* Seperates proteins based on size
* Column has beads that have various pores in them
* allows smaller proteins to enter the pores
* slows down the smaller proteins so they take longer to ellute than the larger ones
* Larger ones will not be slowed by the pores
* This is counter intuitive because the big proteins cant be stick in the pores so they come first - in electrophoresis they come in first and migrate slower
**Affinity chromatopgrahy**
* Many proteins bind to ligands and other molecules
* COlum will contain beads with ligands
* Protein of interest will bind ot the ligand and prevent it from eluting
* protein of interest will be stuck in the cilum
* SOlution of just the ligan will compete with binding for the protein of interest and allow it to ellute
29
Step 4 - detecion from SDS page
* Negative charged SDS the will be loaded into gel and electric current will be applied that oulls proteins through the matrix
* Smaller proteins can easily move through the gel because of theur suze and the bigger ones will not be able to pass
* Crude samples ahve many weight after purifying they have only a single of interes t
30
BCHM218
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