Final Exam Flashcards
1
Q
what is meant by “Living organisms exist in a
dynamic steady state, never at equilibrium with their surroundings”
A
The key concept is that any system is most stable at its lowest free energy state under current conditions. When that state is reached the system is at equilibrium. In a steady state, energy is put into the system constantly in order to maintain a higher free energy state than at equilibrium.
2
Q
dwhy does creating and maintaining order require work and energy (gibbs free energy concepts)
A
Dynamic Steady State is the concept that organisms exist, in many ways, away from what is considered to be the actual chemical equilibrium of certain reactions but that these things are balanced out by cellular conditions that ebb and flow
3
Q
How do non-polar compounds force energetically unfavorable changes in the structure of water (hydrophobic effect)
A
Clustering hydrophobic (nonpolar) regions minimize contact with polar solvent (water) and maximize polar region contact with polar solvent. Is a result of the system achieving the greatest thermodynamic stability
4
Q
how does the hydrophobic effect apply to biomolecular changes
A
The hydrophobic effect stabilized protein structure
Hydrophobic amino acids are pushed to the center of the protein
The hydrophobic effect has entropic significance when it comes to protein folding because as the protein folds, this creates more order, decreasing entropy. In order to counter act that, the entropy is transferred to the surrounding water.
5
Q
what weak interactions are crucial to macromolecule structure and function. Relate pH to structure stability and function.
A
Strong disulfide bonds (covalent) are UNCOMMON in protein structure. Proteins are largely stabilized by
Hydrophobic effect
London dispersion forces
Hydrogen bonds
Electrostatic interaction
disulfide bonds appear in proteins that fold in high temperature because they are not affected by denaturation via heat since their bonds are not dependent on hydrogen interactions
6
Q
how are weak interactions affected by pH
A
pH will cause certain R groups to protonate/deprotonate ( alters the H bonds that can/will form), changing the AA sequence, thus changing function of the protein
7
Q
how does pH relate to structure stability and function of biomolecules
A
pH determines if certain H bonds can occur. In proper pH for a biomolecule it is strong because the H bonds are occuring
in improper pH for a biomolecule it loses its ability to form the right H bonds, it begins to denature, it loses its shape thus losing its function
8
Q
Characteristic pka values for weak acids and bases
A
pKa of carboxyl group: 1.8-3
pKa of amino group: 9-10
pKa of R group: varies
9
Q
how can you use pka values to predict which group predominates at a specific pH
A
calculate the percent fraction using pH= pKa + log [A-]/[HA]
10
Q
how to pka values (acid/ conjugated base) relate to changes in net charge (ionization)
A
amino groups can act as acid or bases due to their amino groups, carboxyl groups, and ionizable R groups (weak acids and bases). Amino acids function as buffers. A zwitterion is a dipolar ion that occurs at neutral pH.
The pH at which the net electric charge is zero is the isoelectric point (pI)
The net charge of the amino acid changes as the pH changes.
pH<pKa>pKa the base form dominates</pKa>
11
Q
what is a zwitterion
A
When the charges on an ion cancel out so that it is neutral
Zwitterions occur at neutral pH
Isoelectric calculations allow you to find the pH at which the given molecule is neutral, i.e. the pH at which a zwitterion is formed
12
Q
what type of bond is formed between amino acids in a polymer
A
amide / peptide bond
amide more for smaller molecules, peptide
13
Q
how do amino acids in a polymer join and how do you read their sequence in a polymer
A
amino acids in a polymer are joined by peptide/amino bonds
amino acid polymers are read from N-terminus to C-terminus
14
Q
what is small oligopeptide isoelectric point
A
pI = pKa1 + pKa2 / 2 for molecule w 2 ionizable R groups
Equation is the same for molecules with more than 2 ionizable R groups, you just have to know which pKa’s to use; the two pKa values that are close to each other
15
Q
what is the meaning of isoelectric point
A
The pH at which the molecule forms a zwitterion, i.e. the pH at which the net electric charge is 0
solubility is at its lowest when the net charge is 0
16
Q
how is isoelectric point used to predict charges when pH changes
A
pH < pKa acid form dominates
pH > pKa base form dominates
LOOK FOR OTHER RELATIONSHIP BETWEEN PI AND PH
The isoelectric point (pI) is the pH value at which the molecule carries no electrical charge
pH>pI: net negative charge
pH<pI: net positive charge
pI= (pK1 + pK2)/2
17
Q
what are the characteristics of secondary structure (alpha helix and beta sheet)
A
Alpha Helix
- R groups face outward (cis)
- 3.6 aa residues per turn
- Right handed
- Intra helical H-bond between amino hydrogen (n) and carbonyl oxygen (n+4) with in the structure work to stabilize it
- Each turn held to adjacent turn by 3-4 H bonds
- Destabilized by proline (Proline will NEVER be found in an alpha helix)
- Alanine is frequently found in alpha helixes because it is small and nonpolar
- like charges can NOT form an alpha helix because they repel and cant form the proper fold/conformation
Beta Sheet
- Zig zag extended conformation
- Single polypeptide
- strongest conformation with right handed turns (I found where the slides said this)
- primarily stabilized by H bonds
- glycine and proline are typically found
- antiparallel sheet is the most stable
- R groups protrude in opposite directions (trans)
18
Q
what determines the type of secondary structure that can be formed by proteins
A
Pattern of H-bonding
^^^^ that is true, BUTTTT
Hydrophobic effect **
19
Q
what forces stabilize secondary, tertiary, and quaternary structure
A
Hydrophobic effect
Hydrogen Bonds
- Secondary level of structure is stabilized by alpha helices and beta sheets
- tertiary and quaternary: stabilized by the r groups in the protein that form hydrogen bonds with each other and other elements of the backbone.
London dispersion forces
Electrostatic interactions
- ** Salt bridges, especially those buried in the hydrophobic environment, strongly stabilize the proteins
20
Q
how are secondary, tertiary, and quaternary structures related to denaturation and Tm
A
- temperature: low temps- protein become too rigid to perform substrate binding but 3D structure is NOT lost. High temps- any weak interaction that is not covalent level primary structure in nature will break with high temp
- pH extremes: causes certain R groups to protonate/deprotonate (alters h-bonds) changing the amino acid sequence, thus function of the protein
- organic solvents: can replace h-bonds in the protein (examples include urea and guanidine). The interaction with organic solvents rather than h-bonds causes denaturation. They also cause denaturing due to the hydrophobic effect, they change the polarity of the environment the protein is in (decreases hydrophobic effect which is really important for protein folding)
- Detergents: detergents have a polar head and nonpolar aliphatic tail which alters the hydrophobic effect. (SDS is a detergent)
Tm: melting temperature is the temperature needed to have 50% of denaturation in a protein. Once a protein has reached its melting temp it can’t refold itself
21
Q
examples of proteins that display tertiary AND quaternary structrure
A
a-Keratin
Collagen
Silk Fibroin
**should we add features of their structure??
22
Q
how do proteins prevent misfolding
A
The native structure is made to be the most thermodynamically favorable
It is more energetically favorable for a chaperone to fix a misfolded protein that to remake a whole protein.
23
Q
Remember the rule: 1° structure determines 2 and 3° structure.
Over all protein shape determines function.
what are the levels of protein structure
A
Primary structure: the peptide bond and protein backbone
Secondary structure: describes the local and spatial arrangement of a segment in a polypeptide. Disregard the positions of R groups interactions with other segments. Stable secondary structures include the alpha helix, the beta conformation, and the beta turn
Tertiary structure: overall spatial arrangement of all atoms in a protein
Quaternary level of structure: spatial arrangement of two or more polypeptide chains to form a functional protein
24
Q
Protein function: using myoglobin and hemoglobin as models to explain protein function: Cooperativity vs no-cooperativity.
A
Cooperativity: binding of a ligand to one site affects the finding properties of a different site on the same protein. Cooperativity can be positive or negative.
- sigmoidal curve
- positive homotropic regulation e.g is cooperativity of oxygen binding
Noncooperativity
- myoglobin
- hyperbolic curve
25
Characteristic biochemical properties of each, related to binding, binding curves, affinity, Kd or p50.
Cooperativity Positive homotropic regulation = increased affinity for substrate as more substrate binds.
Cooperativity Positive heterotrophic regulation = increased affinity for substrate as different ligand binds
Cooperativity = sigmoidal curve
noncooperativity = hyperbolic curve
p50 = partial pressure of oxygen at which half the binding sites are occupied. High p50. Lower p50 = higher affinity
Kd = dissociation constant = how likely it is to dissociate. describes how tightly a ligand binds to a particular protein
26
Understand the effect of Bohr effect, carbon dioxide and 2,3-BPG on hemoglobin function.
Bohr Effect: the pH difference between the lungs and metabolic tissues increases efficiency of O2 transport
- CO2 concentration increases --> blood pH lowered -->reduces affinity for O2 --> greater release of O2 at respiring muscles
*** body safely gets ride of CO2 by converting it to bicarbonate buffer with release of proton, THIS IS PART OF THE BOHR EFFECT
2,3-BPG
- Negative heterotrophic regular of Hemoglobin function
- Stabilizes T-state (low affinity state)
- Small, negatively charged molecules, binds to positive charged central cavity of Hemoglobin
27
Know how to properly use graphical data to answer questions about protein behavior.
this is gonna be thermodynamics/kinetics/michaelis menten/enzyme type stuff. cant really make a card for it but be sure to go over it
28
what are the 5 strategies used by enzymes to increase the rate of reactions
binding substrate = proximity and orientation
stabilization of transition
general A/B catalysis
covalent catalysis
metal ion catalysis
make a separate card for each of the five strategies
29
How does structure relate to enzyme function (think about the strategies for catalysis)
active site of an enzyme is where catalyzed reactions take place. Active sites are holding together key catalytic residues, so that they are positioned just where they need to be to facilitate catalysis (example: active sites of proteases, which brings together an acid, a base and a nucleophile to break covalent bonds on other proteins)
Another important structural role of the active site is recognition. Recognition of what to bind and not bind- recognition of substrate.- A very common mechanism for achieving specificity to substrates, is to simply make other substrates not fit the active site. Enzymes typically have their active site in a hydrophobic pocket at the protein surface, and this pocket is formed so that some substrates fit snugly in it, while others do not
**One of many ways to regulate an enzymes activity is by modulating its structure. Many enzymes can be found in different states, with clear differences in global structure between the states. It can also often be shown that one of these is enzymatically active, and the other not. Often one like to call these two states T and R**
30
Michaelis-Menten equation, definition of each parameter in the equation
V0 = Vmax[S] / Km +[S]
V0 = initial velocity
Vmax = Max velocity
[S] = Substrate concentration
Km = substrate concentration at half vmax
31
Double reciprocal and its application to find Vmax, Km, appVmax and appKm. Conceptual, no calculations required.
Double Reciprocal (Lineweaver Burn) Plot: Enzymes obeying Michaelis mention relationships give a straight line when 1/v0 is plotted against 1/[S]
Y intercept = 1/Vmax
X intercept = -1/Km
Slope = Km/Vmax
appVmax = Vmax/(1+[I]/K1)
Vmax in the presence of an inhibitor
appKm = the experimentally determined variable aKm (i double checked this is correct)
*****double check apparent Km and Vmax
32
Know the different types of enzyme inhibition measured by Michaelis-Menten kinetics (Vmax, Km, slope to determine type of
inhibitor)
make a separate card for each of these
33
Recognize and name carbohydrates by name or linear cyclic structure
I have a card for each of their Fischer projections and each of their haworth projections (i think if I forgot one lmk and I'll add it)
34
products of primary alcohol and aldehyde oxidation and reduction
35
how do carbohydrates form polymers, name of bond
B-glycosidic bonds hold carbohydrates together
36
reducing sugars
a reducing sugar is a substrate at the expense of becoming oxidized (reducing agent)
- for a sugar to function as a reducing agent, the must be an available aldehyde (linear form of sugar reacts because in cyclic form theres no aldehyde present)
- once an aldose participates in a redox reaction the carbohydrate product is an aldonic acid
37
function of polymeric forms of carbohydrates: know names, location, function of polymers discussed
homopolysaccharides
heteropolysaccharides
linear
branched
glycogen
starch (amylose and amylopectin)
cellulose
glycosaminoglycans
heparin and heparan sulfate
glycoconjugates: glycoproteins, glycolipids, proteoglycans
Make a card for each of this
38
recognize and name nitrogenous bases, nucleotides
39
what type of sugar is DNA and what type of sugar is in RNA, what is the difference?
Deoxyribose
Ribose
difference is that deoxyribose is missing an hydroxyl group that ribose is not
40
polymers of nucleotides: how are they formed, how do we read the sequence
polymers of nucleotides are created by phosphodiester bonds
we read AA sequence from 5' to 3'
41
forces that stabilize secondary and tertiary structure of nucleic acids
secondary structure: sequence-dependent side-chain interactions and sequence-independent backbone interactions (particularly hydrogen bonding). hydrophobic effect???
Covalent bonds, hydrogen bonds, and hydrophobic interactions are the forces that stabilize the tertiary structure of a protein
42
recognize and name fatty acids
alpha and omega naming
43
what is the general composition and function of the major lipids discussed in class: triacylglycerol, glycerophospholipids, sphingolipids, and sterols
44
know the physical properties of lipids and how can you use them to explain changes in Tm
Tm = melting temp of DNA (when 50% of it is denatured)
Saturated fatty acids have a higher melting point than unsaturated fatty acids
45
what is the difference between standard gibbs free energy and the actual free energy in cells
46
how can unfavorable reactions take place in cells
Reaction coupling
47
what makes a molecule a high energy molecule
if the molecule has a phosphate group that can be hydrolyzed to release energy that can be used
48
chemical and thermodynamic logics of metabolic pathways: importance of chain reactions (like glycolysis)
Chain reactions make possible unfavorable reactions by using the products of these reactions as reactants in the next reaction in the pathway.
49
how does electron movement generate free energy
when bonds are broken, electrons are released and available to do work
****might need a little more detail
50
how do reduction potentials work
Reduction potentials determine the flow of electrons
electrons flow from systems with negative or lower reduction potential to systems with positive or higher reduction potential
∆G = -nF∆Eº'
^^^ thus, if reduction potential (Eº') is positive, ∆G is negative and favorable
51
what is the functional difference between NAD+ and FAD
52
net equation for all the pathways discussed in lecture
make a separate card for each one
53
what does dehydrogenase mean
54
what is the cellular location for the pathways discussed
make a separate card for each one
55
know the meaning of different enzyme names
56
what reactions represent control points in pathways
57
what is the importance of fermentation
58
how is glycolysis and gluconeogenesis regulated in liver
59
how is the concentration of F2,6-Bisphosphate regulated and the function of this molecule in regulation of glycolysis and gluconeogenesis
60
how is pyruvate converted to acetyl-CoA for entry into the TCA. Know details of the process and how the enzyme is regulated via covalent modification
61
Know the TCA pathway: substrate-enzyme-product
62
what type of reaction is taking place at each step of the TCA
63
importance of specific steps in the pathway
64
what points of control are in the TCA pathway
the citric acid cycle is controlled through the enzymes that break down the reactions that make the first two molecules of NADH.
**Step One: Citrate synthase** is responsible for the rate of reaction in the first step of the cycle when the acetyl-CoA is combined with oxaloacetic acid to form citrate. It is inhibited by high concentrations of ATP, acetyl-CoA, and NADH which indicates an already high level of energy supply. The molecule produced in the reaction, citrate, can also act as an inhibitor of the reaction. Because citrate synthase is inhibited by the final product of the citric acid cycle as ATP, ADP works as an allosteric activator of the enzyme as ATP is formed from ADP. Therefore, the rate of the cycle is reduced when the cell has a high level of ATP.
**Step Three**: The enzyme** isocitrate dehydrogenase** is an important catalyst in the third step of the reaction. It regulates the speed at which the citrate isomer isocitrate loses a carbon to form the five-carbon molecule α-ketoglutarate. The coenzyme NADH is a product of the reaction and, at high levels, acts as an inhibitor by directly displacing the NAD+ molecules it is formed from.
**Step 4: The enzyme α-ketoglutarate dehydrogenase** is another important catalyst in the fourth step of the cycle where α-ketoglutarate also loses a carbon and combines with Coenzyme A to form succinyl CoA. The two products of the reaction, succinyl CoA and NADH, both work as inhibitors at large concentrations.
The 3 forms of control are: substrate concentration, product inhibition, and feedback inhibition
65
What points of control are in the glycolysis pathway
Steps 1,3, and 10
Step 1: conversion of glucose to glucose-6-phosphate. Catalyzed by hexokinase.
Step 3: Conversion of Fructose-6-Phosphate into Fructose-1,6-Bisphosphate. Catalyzed by PFK-1
Step 10: Conversion of PEP to pyruvate
66
Why is the TCA known as cellular respiration
The combo of glycolysis, the citric acid cycle, and the electron transport chain are known as cellular respiration.
It’s because cell seems to “respire” in a way that it takes in molecular oxygen (as an electron acceptor) and releases carbon dioxide (as an end product).
67
what is the function of O2 in cellular respiration
- In cellular respiration oxygen acts the final electron acceptor for electrons removed from intermediate compounds in glucose metabolism
- in the citric acid cycle: the NADH and FADH2 produced must transfer their electrons to the next pathway in the system, which will use oxygen. If oxygen is not present, this transfer does not occur. The citric acid cycle does NOT occur in anaerobic respiration.
68
What is the rate of absorption of nutrients determined by
The cell's surface area
69
As a cell grows larger, its volume _____ faster than its surface area
As a cell grows larger, its volume increases faster than its surface area
70
High surface are to volume ratio allows cells to do what quickly
move material in and out of the cell
71
Cells of the intestinal lining have small protrusions known as microvilli that increase cellular surface area. What purpose might that serve?
to increase absorbtion
72
select the option that best describes a chemical equilibrium:
- All of the options represent characteristics of chemical equilibrium
- Temperature remains constant when the system is at equilibrium
- Concentration of reactants and products remain constant when the system is at equilibrium
- Volume remains constant when the system is at equilibrium
all of the options represent characteristics of chemical equilibrium
73
Which of the following statements about equilibrium is FALSE?
- At equilibrium, there is no net change in the system
- At equilibrium, the concentration of reactants and products remains the same
- At equilibrium, the forward and backwards reactions cease to occur
At equilibrium, the forward and backwards reactions cease to occur
74
What can the relation of Keq and Q tell us about where equilibrium lies
Q< Keq: reaction proceeds toward products
Q > Keq: reaction favors reactants
Q = Keq: no particular side is favorable; reaction is easily reversible
75
What do Keq values tell us about where equilibrium lies
Keq = 1: equilibrium
Keq > 1: products are favored
Keq < 1: substrate is favored
76
Consider the binding of oxygen to hemoglobin (protein without oxygen): Hb + O2 --> Hb-O2. If the pressure of the system decreases, predict what is the effect on the equilibrium shown?
The equilibrium shifts to the left
77
Definition of:
open system
closed system
isolated system
open system: system exchanges both heat and energy with its surroundings
closed system: system exchanges energy but not matter with its surroundings
isolated system: system does not exchange matter or energy with its surroundings
78
what does entropy (S) measure
thermodynamic measurement of molecular randomness and disorder
positive delta S: favorable
negative delta S: external input of energy needed for reaction to proceed
delta s alone is not enough to determine the spontaneity of a reaction
79
the change in enthalpy of a system is due to heat supplied at
constant pressure
80
A system that increases in temperature at constant pressure is
gaining energy from its surroundings
81
what are the cellular dimensions of a cell limited by? how do parameters like volume, surface area, and size affect diffusion rate?
cellular size is limited by rate of diffusion of nutrients and waste
- upper limit of the cell is limited by rate of transport and need to deliver O2 to all parts of the cell
- lower limit of cell is determined by number of molecules needed to sustain living state of the cell
- larger surface area to volume ratio results in faster rates of diffusion
82
What is enthalpy (H) a measure of
heat content, roughly reflecting # and kinds of chemical, state function
positive delta H: final state has a greater amount of PE
negative delta H: final state has less PE than starting materials --> Energy is released to surroundings --> favors forward direction
83
know the terminology and be able to identify structures that are: configurational isomer, conformational forms, enantiomers, diastereomers, chiral centers
- conformation: spatial arrangement of substituent groups that are free to assume different positions in space
- geometrical isomers (or cis trans isomers) differ in the arrangement of substituents groups with respect to the double bond
- chiral centers are asymmetric carbons. A molecule can have 2^n stereoisomers (n is the number of chiral centers)
- enantiomers: stereoisomers that are mirror images of each other. These have nearly identical chemical reactivities but differ in optical activity
- diastereomers: stereoisomers that are not mirror images of each other
84
why is molecular spatial distribution important in biomolecules?
spatial distribution influences molecules interactions and physical interplay with its surroundings.
85
why are thermodynamics an important aspect of biochemistry?
it deals with the energy output of chemical reactions and helps us understand how energy flows in biological cells, how large molecules assemble into complex structures, etc.
86
what is the first law of thermodynamics?
In any physical or chemical change, the total amount of energy in the universe remains constant, although the form of the energy may change
87
what is the second law of thermodynamics
the randomness in the universe is constantly increasing
88
what is Gibbs free energy? what sign determines if it is favorable or unfavorable? what is the formula for gibbs free energy? under what conditions (s, h, and t) is gibbs free energy favored and not favored?
Gibbs free energy the amount of energy available to do work. It relates to the change in enthalpy and entropy that take place during a reaction at a specific temperature. A positive delta G is unfavorable. A negative delta G is favorable. When delta G=0 a system is at equilibrium.
Delta s (+), Delta H (+):
Delta s (-), Delta H (+):
Delta s (-), Delta H (-):
Delat s (+), Delta H (-):
89
what is le chateliers principle and how does this apply to biochem reactions
When a system is at equilibrium if an external agent disrupts the equilibrium, the system will spontaneously move in the direction that diminishes the disruption. This maintains the equilibrium without having to add additional energy to the system. For unfavorable reactions we can couple the reaction or alter concentration to make it favor the direction of the products.
90
what is the mass-action ratio? how is it calculated and what does it mean in terms of Keq and delta G
The mass-action ratio (Q) is the ratio of product concentrations to reactant concentrations at a given time. This can be calculated to tell how far the reaction is from equilibrium.
91
what is the formula for standard free energy change
delta G= delta G (standard condition) +RTlnKeq
92
what are pathways? how does this relate to catabolism and anabolism? what is catabolism and anabolism?
Pathways are sequences of consecutive reactions in which the product of one reaction becomes the reactant in the next.
Catabolism is degradative, free energy- yielding reactions. This drives ATP synthesis and produces the reduced electron carriers NAD(P)H
anabolism are synthetic pathways that require the input of energy. They take simple matter and create complex matter. The energy input used comes from catabolic pathways
93
what is metabolism and the what is the unity of life
Metabolism is the overall network of enzyme-catalyzed pathways, both catabolic and anabolic.
Unity of life: pathways of enzyme-catalyzed reactions that act on the main constituents of cells and its nearly identical in all living organisms
94
what are hydrogen bonds? why are they biologically relevant?
weak electrostatic attraction between one electronegative atom and a hydrogen atom covalently linked to a second electronegative atom. Hydrogen bonds are found within the structure of DNA molecules. They are main reason for the folded shape structure for enzymes that allows biological reactions to occur.
they are individually weak but collectively strong interactions
95
why is polarity and solubility so important in biochemistry?
polarity allows you to predict whether or not a solute will be soluble to a solvent (like dissolves like)
96
what is the hydrophobic effect? why is this so important to our proteins
the tendency of nonpolar substances to aggregate in aqueous solutions and exclude water molecules. It is one of the major driving forces behind the proper folding of proteins
97
what does pka measure and what is its formula
pKa measures acidity. pKa= -logKa
97
what amino acids are essential?
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine
98
classification of each amino acid based on r group
nonpolar aliphatic: glycine, alanine, proline, valine, leucine, isoleucine, methionine
Aromatic R groups: phenylalanine, tyrosine, tryptophan
Polar, uncharged R groups: serine, threonine, cysteine, asparagine, glutamine
Positively charged R groups: lysine, histidine, arginine
Negatively charged R groups: aspartate and glutamate
99
what types of modifications lead to uncommon amino acids? how do these modifications occur?
they can be modified after protein synthesis, during protein synthesis, or modified transiently to change a proteins function (e.g. phosphorylation.
Free metabolites
100
why is primary structure of a protein important
Drives the folding and intramolecular bonding of the linear amino acid chain, which ultimately determines the protein's unique three-dimensional shape. shape determines function
101
what is the chemical nature of a polypeptide bond and how does it influence protein folding
covalent bonds between alpha carbons. The are three types of covalent bonds alpha C-N, alpha C-C, and C-N. The C-N bonds have partial double bond character so they can't rotate freely (resonance). The resonance forces the peptide bond to conform within a plane
102
why does secondary level of structure have such low diversity?
the C-N bonds force the peptide backbone to have a planar structure
103
true or false: Temperature does not affect the ionization constant of a molecule
false
104
Which component is absolutely necessary for the purification of a protein?
A) column chromatography
B) the gene sequence of the protein
C) a means of detecting the protein
D) a centrifuge
a means of detecting the protein
105
denaturing gel electrophoresis separates proteins based on differences in:
A ) size and shape
B) molecular mass
C) charge
D) amino acid content
size and shape
106
What measurement increases during purification of an enzyme:
A) activity
B) total protein
C) specific activity
D) fraction volume
specific activity
specific activity is the number of enzyme units per mg of total protein. The specific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure
107
what is the highest level of protein structure in human insulin, which has two polypeptides of different mass linked by several disulfide bonds?
A) primary
B) secondary
C) tertiary
D) quaternary
quaternary
when a protein has 2+ polypeptide subunits, their arrangement in space is referred to as quaternary structure
108
which statement is true about mass spec?
A) mass spec can be performed on analytes in the liquid phase
B) mass spec can obtain the sequences of multiple polypeptide segments of 100 residues each
C) the mass (m) of an analyte is used to deduce the mass to charge ratio with high precision
D) MALDI MS requires treatment of proteins with a protease before injection into a mass spec
E) mass spec can monitor changes in the cellular proteome as a function of metabolic state
mass spec can monitor changes in the cellular proteome as a function of metabolic state
- when coupled to peptide separation protocols, mass spec can document a complete cellular proteome. Changes in the cellular proteome can be monitored as a function of metabolic state or environmental conditions
109
Which statements are true about the formation of a peptide bond:
A) a peptide bond forms when the carboxylic acid groups, R-COOH, of the two amino acids react together
B) a molecule of water links the amino acids together during peptide bond formation
C) amino groups amino acids act as electrophiles to remove the OH from alpha carboxyl groups
D) the amino group of one amino acid works as a nucleophile to displace the hydroxyl group of another amino acid
E) an OH group is removed from the alpha carboxyl group of one amino acid and an H from the alpha amino group of another amino acid
D) the amino group of one amino acid works as a nucleophile to displace the hydroxyl group of another amino acid
E) an OH group is removed from the alpha carboxyl group of one amino acid and an H from the alpha amino group of another amino acid
110
how does mass spectrometry work?
atoms or molecules are ionized using a high-energy electron beam and then separated based on their mass-to-charge ratios (m/z).
111
what information can you gain from mass spectrometry and tandem mass spectrometry
Mass spectrometry provides information concerning the molecular structure, atomic mass of whole molecules, molecular fragments, and atoms.
tandem ms: two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. First round sorts peptides produced by cleavage, second step, second round measures m/z ratios of charged fragments.
112
how are samples introduced for mass analysis?
Step 1: ionize analytes into a vacuum
Step 2: introduce charged molecules to electric and/or magnetic fields
Step 3: charged molecules move through field as function of the mass to charge ratio, m/z
Step 4: deduce mass of analyte
113
what are the limitations of tandem MS?
cannot be applied for single-cell analyses as it is insensitive to analyze such small amounts of a cell
114
what is the chemical nature of a polypeptide bond and how does it influence protein folding
covalent bonds between alpha carbons. The are three types of covalent bonds alpha C-N, alpha C-C, and C-N. The C-N bonds have partial double bond character so they can't rotate freely (resonance). The resonance forces the peptide bond to conform within a plane
115
why does secondary level of structure have such low diversity?
the C-N bonds force the peptide backbone to have a planar structure
116
size-exclusion chromatography
- SEC separates molecules by differences in size as they pass through a resin packed in a column
SEC resins consist of a porous matrix of beads that lack reactivity and adsorptive properties. After sample has entered the column, molecules larger than the pores are unable to diffuse into the beads, so they elute first. Molecules that range in size between the very big and very small can penetrate the pores to varying degrees based on their size. If a molecule is smaller than the smallest of the pores in the resin, it will be able to enter the total pore volume. Molecules that enter the total pore volume are eluted last. Samples are eluted isocratically so there is no need to use different buffers during the separation.
117
ion exchange chromatography
- Ion exchange chromatography (IEC) is fundamentally a separation method which uses net charge of proteins for their separation and purification.
- The net surface charge of proteins varies according to the surrounding pH. Above its isoelectric point (pI), a protein will bind to a positively charged anion exchanger. Below its pI, a protein will bind to a negatively charged cation exchanger.
- Proteins bind as they are loaded onto a column at low ionic strength. The conditions are then altered so that bound substances are desorbed differentially. Elution is usually performed by increasing salt concentration or changing pH in a gradient
118
affinity chromatography
- In affinity chromatography (the target protein is specifically and reversibly bound by a complementary binding substance (ligand). The sample is applied under conditions that favor specific binding to the ligand. Unbound material is washed out of the column.
- The bound target protein is recovered by changing conditions to those favoring elution. Elution is performed specifically, using a competitive ligand, or nonspecifically, by changing, for example, pH, ionic strength, or polarity. The target protein is eluted in a purified and concentrated form.
- Because of its high selectivity, affinity chromatography can be used for single-step purifications. More commonly, however, AC is used as the first capture step, followed by one or more polishing steps to remove remaining impurities.
119
SDS-PAGE
- SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight.
- The first thing to know about how SDS-PAGE works is that it separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field.
-The movement of any charged species through an electric field is determined by its net charge, its molecular radius, and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge nor their molecular radius is molecular weight dependent.
- Instead, their net charge is determined by amino acid composition, i.e. the sum of the positive and negative amino acids in the protein and molecular radius by the protein’s tertiary structure
-So in their native state, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape.
- To separate proteins in an electrical field based on their molecular weight only, we need to destroy the tertiary structure by reducing the protein to a linear molecule, and somehow mask the intrinsic net charge of the protein.
-SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent, it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules.
- **Since the SDS-coated proteins have the same charge-to-mass ratio, there will be no differential migration based on charge**
120
gel-electrophoresis
- Gel electrophoresis is a technique used to separate DNA fragments according to their size.
- DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel.
- DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
-mWhen a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.
121
what are the 20 amino acids (structure, name, and one letter code)
122
what amino acid is this
alanine (A)
123
what amino acid is this
glycine (G)
124
what amino acid is this
isoleucine (I)
125
what amino acid is this
Leucine (L)
126
what amino acid is this
proline (P)
127
what amino acid is this
valine (V)
128
what is this amino acid
phenylalanine (F)
129
what amino acid is this
tryptophan (W)
130
what amino acid is this
aspartate/ aspartic acid (D)
contains a negative charge
131
what amino acid is this
glutamate/ glutamic acid (E)
has a negative charge
132
what amino acid is this
arginine (R)
- has a positive charge
133
what amino acid is this
histidine (H)
- has a positive charge
134
what amino acid is this
lysine (K)
- has a positive charge
135
what amino acid is this
serine (S)
136
What amino acid is
Threonine (T)
137
what amino acid is this
Cysteine (C)
138
what amino acid is this
methionine (M)
139
what amino acid is this
asparagine (N)
140
what amino acid this
glutamine (Q)
141
what amino acid is this
Tyrosine (Y)
142
what amino acid is this
alanine (A)
143
what amino acid is this
glycine (G)
144
What amino acid is this
Leucine (L)
145
what amino acid is this
Valine (V)
146
what amino acid is this
aspartate (D)
147
what amino acid is this
Glutamate (E)
148
what amino acid is this
isoleucine (I)
149
what amino acid is this
arginine (R)
150
what amino acid is this
lysine (K)
151
what amino acid is this
serine (S)
152
what amino acid is this
threonine (T)
153
what amino acid is this
cysteine (C)
154
what amino acid is this
methionine (m)
155
what amino acid is this
asparagine (N)
156
what carbohydrate is this
D-glyceraldehyde
157
what carbohydrate is this
D-erythrose
158
what carbohydrate is this
Dihydroxyacetone
159
what carbohydrate is this
D-ribose
160
what carbohydrate is this
D-Ribose
161
what carbohydrate is this
D-arabinose
162
what carbohydrate is this
alpha D-arabinose
163
what carbohydrate is this
D-xylose
164
what carbohydrate is this
alpha D-xylose
165
what carbohydrate is this
D-glucose
166
what carbohydrate is this
Glucose
167
what carbohydrate is this
D-mannose
168
what carbohydrate is this
D-mannose
169
what carbohydrate is this
D-galactose
170
what carbohydrate is this
Galactose
171
what carbohydrate is this
D-Erythrulose
172
what carbohydrate is this
D-ribulose
173
what carbohydrate is this
alpha D-ribulofuranose
174
what carbohydrate is this
D-xylulose
175
what carbohydrate is this
alpha D-xylulofuranose
176
what carbohydrate is this
fructose
177
what carbohydrate is this
fructose
178
Vmax is ___
the fastest an enzyme can produce product in a set amount of time
179
3 conditions for Michaelis menten equation
1) Dynamic Steady State
2) V0 would be dependent on the rate limiting step of the reaciton and the concentration of the enzyme substrate complex
3) concentration of the enzyme for each assay needed to be exactly the same otherwise there would be an influence on initial velocity
180
