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BCHM218 > Mod1 real > Flashcards

Mod1 real Flashcards

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
    *
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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
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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
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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]
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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
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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
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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

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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

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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
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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
    *
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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
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13
Q

What are salt bridges

A
  • Involve 2 interactions
  • H and eletcrostatic
  • Happens between eletcrically charged amino acidic and basic groups
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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
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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
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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
Q

What are the general steps of protein purification

A
  • Cell lysing - protein extraction and breaking apart the cell
  • Centrifugation - seperating density of proteins
  • fractionation - seperating the types of proteins
  • Protein detection
26
Q

Step 1 Cell lysis

A
  • 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
Q

Step 2 Centrifugation

A
  • 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
Q

Step 3 Fractionation

A

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
Q

Step 4 - detecion from SDS page

A
  • 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
Q
A

BCHM218 (3 decks)

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