Quiz 1 Material - Intro, Zaia lectures, Hemoglobin/myoglobin Flashcards
Ameloblast
Cell Type That Creates the Enamel
Odontoblast
Forms Dentin, and Found in Pulp
Dentin Sialophosphoprotein (DSPP)
Component in Formation of Dentin,
Gene is seen mutated in Dentinogenesis Imperfecta II and III as well as Dentin Dysplasia II
Anton Van Leeuwenhoek
Coined the Term ‘animalcules’ and observed oral bacteria
Robert Hooke
Coined the word ‘cells’
Matthias Schleiden
Stated all plants are made of cells and come from one cell
Theodor Schwann
Worked with Matthias Schleiden and proposed three tenants of the cell theory
- all organisms are composed of one or more cell
- the cell is the smallest unit of life
- cells can come from inorganic materials
Rudolf Virchow
Fixes Schleiden and Schwanns third point by saying that cells can only come from previous cells.
Properties of the Archaea
Transcription looks like that of eukaryote, but DNA looks like that of a prokaryote
Steps in Endosymbiotic Theory
-Large anaerobic Host Took up Small aerobic bacteria
- Large bacteria protects and provides nutrients to small one
- small bacteria generates additional energy for larger bacteria
small bacteria loses ability to proliferate independently
Konstantin Mereschkowski
Proposed chloroplasts were distant relative to photosynthetic bacteria
Lynn Margulis
Furthered Mereschkowski’s theory and was all over the endosymbiotic theory.
Evidence to Support Endosymbiotic Theory (6)
- mitochondria and chloroplasts can only arise from pre-existing mitochondria and chloroplasts
- mitochondria and chloroplasts have their own genome that looks like prokaryotes
- both genomes consist of circular DNA with no histones
- Mito and Chloro have their own protein assembly machinery
- antibiotics that black protein synthesis in bacteria also block protein synthesis in mito and chloro
- inhibitors of protein synthesis in eukaryotes dont have an effect on bacterial proteins
Jared Hara
Has Leber’s Hereditary Optic Neruopathy which is a mitochondrial disease that results in blindness
Which came first plant or animal Cells
The animal cell because it only contains mitochondria, while plant has chloro and mito
Features in common between prokaryotes and eukaryotes (5)
- Genetic information in DNA
- The DNA uses common code (ACTG)
- Mechanism of transcription and translation are strikingly similar
- Metabolic pathways are conserved
- chemical energy is stored as ATP
Examining Phylogenetic Tree Reveals (3)
- Life Requires Energy and must conver to useful work
- Biochemical reactions need a catalyst
- life depends on info encoded in genes
Watson and Crick
Identified the Double Helix using X-Ray Crystallography
Rosalind Franklin and Maurice Wilkins
Helped with Double Helix Structure determination with Watson and Crick
How Many Base Pairs are in the Human Genome
3 billion
How many protein coding genes are there
20,000
What is the central Dogma of molecular Biology
DNA -> DNA - Replication
DNA -> RNA - Transcription
DNA -> Protein - Translation
Dentinogenesis Imperfecta (DI) Phenotype
- Discolored Teeth
- Poorly Mineralized Dentin
- Fractured Enamel
- Rapid Wear and Attrition of teeth
Dentinogenesis Imperfecta Types and Cause
I - mutation in Collagen gene, and usually associated with Osteogenesis Imperfecta (OI)
II - Have a mutation in the DSPP Gene
III - Aka Brandywine Isolate, and also have mutation in DSPP gene
Dentin Defects (4)
Dentinogenesis Imperfecta
Dentin Dysplasia (DD)
Hypophasia
Ehlers Danlos Syndrom (ED)
Dentin Dysplasia Type II Cause
Has a DSPP mutation
Amelogenesis Imperfecta (AI) Phenotype
Thin Enamel
Hypomineralization (not enough mineral)
Enamel Abrades Easily
Very Sensitive Teeth
Amelogenesis Imperfecta (AI) Mutations
AMELX - amelogenin ENAM - Enamelin MMP20 KLK4 FAM83H
Hypodontia (Cause and Effect)
Cengenital Missing teeth caused by disfunction in MSX1 and PAX9 transcription factor problems
Vitamin D Purpose (3)
Calcium absorption
Mineral Homeostatis
Bone Health
Bisphenol A (BPA) effects
Hypomineralization
Vitamin D Deficiency Effects
Low Mineral bone density (osteopenia and osteoporosis)
Infectious disease and inflammatory disease
cancer
Periodontal disease and tooth loss
Poor birth outcomes
Alzheimer, Schizophrenia and depression
Cardiovascular disease
Stages of Periodontal Disease
Norma, Gingivitis, Periodontitis, Advanced Periodontitis
Periodontal Disease Causes
Bacteria are not actually a good indicator as each host reacts differently.
Environment factors are actually huge
and inflammation of periodontium is very common.
Tumor Necrosis Factor Alpha (TNF α)
pro inflammatory cytokine leading to inflammation seen in the relation between obesity and periodontal disease. TNF suppresses insulin action
PubMed
Database of Scientific Papers
OMiM
Database of human genes and genetic disorders
Cn3D
Database to see the three dimensional structures of molecule
First Law of Thermodynamics
Energy is not created nor destroyed and constant in a system
Second Law of Thermodynamics
Entropy always increases
Gibbs Free Energy Equation
ΔG = ΔH - T ΔS H = Chemical Bond Energy S = Entropy
ΔH when negative
Negative when reactants have more energy than products (gives off energy)
ΔS when it is positive
Is positive when when more disorder
When is ΔG always Spontaneous
When ΔH is negative (gives off energy) and ΔS is positive (more disorder)
How ΔS and heat transferred relate
When ΔS increases than the heat transferred out goes up
How Temperature and ΔS are related
When Temperature goes up then it is hard to transfer heat out so the ΔS goes down
What determines the reaction speed
Activation Energy, and not the Spontaneity
How many water molecules can H-Bond to a water
4
Van Der Waals’ Force Definition
A weak force created by the transient nature of electron clouds when molecules nearby naturally attract
Driving Force of Hydophobic Interaction
The Disorder increases for water and therefore it is driven by the entropy of water.
Kw Equation
Kw = [H+]{OH-]
pH Equation
pH = -log[H+]
Ka Equation
Ka = [H+][A-]/[HA]
What is a pKa
The pH when the reactants are half dissociated, and it is the - log of the Ka
Henderson Hasselbach Equation
pH = pKa + log [A-]/[HA]
Result of Fluoride Treatment
Hydroxylapatite converts to fluorapatite and impedes demineralization
How to Calculate pI
Average the two pKas flanking the new neutral form of the peptide chain, this will be the net neutral pH (Practice this)
pKa of terminal Carboxyl Group
~3
pKa of terminal amino group
~8
Hydroxyapatite Structure
Calcium, phosphate and OH, interacts with protein for shape, and has a woven structure
What Configuration are Amino Acids in
L-Configuration
Glycine (chirality, when seen, collagen)
Gly, G
achiral, Small, nonpolar, neutral
in the middle of hydrophobicity and hydrophillic
seen when need space and flexibility
because of collagens form of a triple helix there must be a glycine every 3 residues
Alanine
Ala, A
Small Neutral and nonpolar, and hydrophobic
Valine
Val, V
Neutral, nonpolar, hydrophobic
seen interior of proteins
Leucine
Leu, L
Neutral Nonpolar hydrophobic
seen in interior of protein
Isoleucine
Ile, I
nonpolar and hydrophobic
Has a chiral Beta Carbon in R Group
Methionine
Met, M
Non-polar and hydrophobic
Rare
easily oxidized to sulfoxide and sulfone
Phenylalanine
Phe, F
aromatic, very non-polar, hydrophobic
Tyrosine
Tyr, Y
Aromatic, hydrophobic and phillic grey area, polar and non polar
absorbs at 280nm
phosphorylates at the OH
Tryptophan
Trp, W aromatic, very large, and hydrophobic, and nonpolar rare absorbs at 280nm only AA that fluoresces
Serine
Ser, S
Polar and hydrophillic,
phosphorylates and O-glycosylates post translationally
Threonine
Thr, T
Polar, more hydrophobic
has chiral side chain
post translationally modifies by phosphorylation and o-glycosylation
Cysteine
Cys, C
has a SH group and is super reactive
oxidizes in presence of oxygen
nucelophillic
must be alkylated when sequencing and analyzing
reacts in metal reactions and is seen in metalloproteins
oxidize to disulfide bonds
forms parts of antioxidants such as glutothione
Arginine
Arg, R
Positively Charged base,
hydrophillic polar
Lysine
Positive charged and basic
hydrophillic and polar
Histidine
His, H
Neutral and polar but basic
HYDROPHOBIC grey area
participates in acid base reactions
Aspartate
Asp, D
Acidic negative charged, hydrophilic and polar
can esterify to glutathion
Glutamate
Glu, E
Acidic negative charge, very polar
can have esterification
Asparagine
Asn, N
polar, and hydrophillic
can deaminate to aspartate,
does not give or remove H
Glutamine
Gln, Q
Polar and hydrophiliic
does not give or remove H,
can deaminate to glutamate
Proline
Pro, P is an imino Acid no rotation around the N and alpha carbon no resonance of amide bond usually cis, breaks helices
Aliphatic
Means it does not have a ring
Non-Polar Amino Acids
Gly, Ala, Val, Leu, Ile, Phe, Pro (some others are in the grey area)
Hydrophobic amino Acids
Ala, Val, Leu, Ile, Met, phe, Tyr, Trp His Thr
Polar Amino Acids
Ser, Thre, Csy, Asn, Gln, Lys, Arg, his Asp Glu Tyr (has some polar character)
Hydrophilic Amino Acids
Arg, Glu, Asp, Lys, Pro. Cys, Ser, Asn, Gln
Charged Amino Acids
Asp, Glu, Lys, Arg
Basic Amino Acids
Lys, Arg, His
Acidic Amino Acids
Asp, Glu
Phi and Psi Angles
The two bonds around the Alpha Carbon rotate in specific ways, and if you know these angles for all bonds you will know the three d structures. The phi bond is the N-C bond and psi angle is the C–C
Hydrogen Bonding in Alpha Helix
Between every N and C=O every 3.6 residues.
The pitch of an Alpha Helix
of Angstroms that rises per turn
Amino Acids not found in Alpha Helix
branches (Val Thr Ile,) H-Bonding donating (Ser, Asp, Asn). Do See Glu, and Gln
Amino Acids found in Beta Strands
Val, Ile, Phe, Tyr, Trp, thr. Tolerates large R groups
Proline breaks strands
H Bonds in Parallel vs Antiparellel Beta Strands
Straight H-bonds in antiparallel, making more stable (seen on outside of proteins)
Diagonal bonds in parallel, making less stable and seen on inside of proteins
Chaperones (examples)
Stabilize proteins during folding.
Use ATP (folding on own does not)
GroES and GroEL examples
Mucin
long polypeptide that is carbohydrate modified and coats mucosal surfaces
Non-newtonian Fluid
Viscosity changes with pressure
Ex// paint ketchup and saliva
Caused y proteins in the fluid
Differential Centrifugation
step by step centrifugation and removing of supernatant to isolate parts (organelles) of the cells
Dialysis
Size based separation in which the volume is kept constant
Centrifugal Concetrators
centrifugal force will push solution through the dialysis pore, which then decreases the volume
Gel-filtration Chromatography
Aka size exclusion chromatography,
beads have small pores in side that small proteins get stuck in so large molecules will elute first
what absorbance do peptides have
220nm
Ultracentrifugation
forms a sucrose density gradient and then spin the protein down it. The proteins will seperate by S value, which is the sedimentation coefficient, a value representing the density and shape
Ion Exchange Chromatography
Use beads with charged attachments to catch proteins based on pI
Can change the pH to trap certain proteins
use salt to elute
Cation Exchange
The positive proteins (cations) will get stuck to a negative beed
Anion Exchange
Beads are positive and capture the anion (negative protein)
Affinity Chromatography
Beads have very specific attachments like an antibody, which will catch whichever protein you want
to elute you can add a competitor or denature
SDS -PAGE
based on size.
protein is put through detergent and coated in negative charge
agarose is dense and so bigger proteins will move slow, and small peptides will move quick
Reading SDS -PAGE
Position shows MW
Size shows amount
and if there are many bands the protein is not pure
Anode and Cathode
Anode is positive charged so anions move toward it
Cathode is negative charge and so cations move toward it. in SDS page the anode is at the bottom
Two-Dimensional Gel Electrophoresis (and Advantages and Disadvantages)
Separate by pI and then MW dimension
Advantages - see multiple proteins and patter
Disadvantage- post translational modifications ruin spots, bad resolution and non reprorducable
Fluorescamine
After dissolving peptide in acid then react with this in order to fluoresce the amino acids
with this you can look at peaks
Amino Acid Analysis
Using Fluorescamine you need a lot of protein and you can compare theoretical and experimental. process is dissolve the peptide and chromotraphy it to seperate and then fluoresce and compare to see if it is diseased
Edman Degredation (advantage and disadvantage, and two molecules involved)
Used to sequence one AA at a time
phenylisothiocyanate - reacts with n terminus of chain
phenylthiohydantoin clips off AA at the end
advantage - can do multiple mixes and is accurate
disadvantage - its really slow and mass spec is better
High Performance Liquid Chromatography (HPLC)
Make beads super small. Must apply pressure actually to get the peptides through but the result is really specific separation.
Reverse Phase HPLC
A specific type of HPLC in which one adds hydrophobic groups to beads. Polar molecules flow through first and an increasing organic solvent gradient is used to elute by hydrophobicity
Liquid Chromatography-mass spectrometry
using the elution from a chromatography then mass spectrometry in order to get peptide sequences
ELISA - Enzyme linked immunosorbent Assay
To show amount of protein using antibodies
Indirect ELISA
Fix protein, use specific antibody, then a general antibody that can be detected
This is challenging because you can get non-specific binding and one needs a good specific antibody
Sandwhich ELISA
More specific then indirect, but also requires good antibodies. You fix one antibody, then put in the protein to bind. One then tag the protein with another antibody on the other side that can be detected
Western Blotting
After SDS-PAGE one will transfer the blot to a sheet using electricity. One then uses an antibody to only illuminate the band in question. This can help in comparing phenotypes of diseases
Circular dichroism
Caused by differences in light absorption between clockwise and counterclockwise vectors of light. Spectrums are created that are specific to proteins and their structure
can compare proteins phenotype using
NMR Spectroscopy
Magnetizes the molecules and results in spinning of the hydrogen atoms. This data can be analyzed to figure out structure.
X-Ray Crystallography
Pass X-Ray through the a crystallized protein and observe its diffraction. The x-ray will scatter (due to interactions with electron clouds) and professionals can read this information to determine structure.
Trypsin Digest
When you digest a peptide with Trypsin it reliables cleaves at Arg and Lys
Compare and Contrast Myoglobin, and Hemoglobin
Similar - in Structural motifs, carry oxygen, have prosthetic group
Contrast - AA sequence is less than 25% similar and hemoglobin is a quaternary protein
Which are the crucial Histidines in hemoglobin
F8 and E7
Myoglobin Properties
Located in muscle
Compact with helical sections
only charges are located on the two histidines
What is the prosthetic group in myoglobin and hemoglobin?
Ferroprotoporphyrin
Methemoglobin
If oxygen oxidizes the ferrous to the ferric in the heme it will no longer coordinate with oxygen
Histidine E7 (what it does and what happens when mutated)
Is the distal histidine that donates H-Bond to the oxygen but also forces bent confirmation
Blocks oxidation of the ferrous
with mutation you get a lot of methemooglobin
Ferrous Ion (How Does it Bind)
Binds to 6 ligands. Four of which are Nitrogen in the porphyrin ring. 2 are perpendicular to the ring.
Carbon Monoxides effect on hemoglobin
CO binds more strongly to iron then oxygen. Because of histidines though you can actually has some CO in the body as it will be blocked and oxygen in high concentration can overpower it
Histidine F8
Binds directly to the Iron
Hemoglobin A
Found in adults. Has 2 alpha and 2 Beta subunits
Mechanism for Cooperativaty
- The T (Deoxy) form has its Iron out of the plane porphrin plane
- O2 enters and pulls electrons from the ferrous
- this lowers it in to the plane
- an actual conformational change happens rotating 15 degrees counterclockwise
- thus opening the molecule for more cooperation
2,3 - diphosphoglycerate affect on hemoglobin
Stabilizes deoxyhemoglobin, thus weakening affinity for oxygen
H+ ions and CO2 effect on hemoglobin
promotes release of oxygen, lower binding affinity by stabalizing deoxy form
Bisphosphoglycerate - BPG
- Has a minus five charge
- Stabalizes the T state (Deoxyhemoglobin)
- charges interact with positive charges in beta chain
- BPG moves affinity curve right
- oxygen must knock BPG out it get in to the hemoglobin (seen in lungs)
Hemoglobin F
Fetal Hemoglobin containing 2 alpha and 2 gamma subunits
BPG in Fetuses
Does not exist due to the lack of Beta chain.
Histidine is replaced by Serine in the Gamma subunit and therefore less affinity to the negative BPG
This makes deoxy less stable and affinity curve moves left for oxygen
allows for oxygen to flow from mother to child
Bohr Effect
Decreasing pH shifts curve right and creates a lower affinity for oxygen
Mechanism of Bohr Effect
- When the pH goes down the histidine becomes protonated (His-146)
- His-146 ion pairs with Asp-94
- By forming this it pulls His-f8 which then pulls on the iron
- this removing it from the plane and thus unloading the oxygen
- this stabilizes the deoxy form
The Two Ways Carbon Dioxide lowers Oxygen Affinity
1) Co2 becomes bicarb thus releasing protons
2) co2 reacts with the n-terminus amino to form carbamates - which stabilize salt bridges in the T Deoxy state
Haldane Effect
Deoxygenation of blood increases its ability to carry co2
Carbonic Anhydrase
Converts Carbon dioxide to bicarbonate and protons. Thus if you were to inhibit this it would shift an affinity curve left for oxygen.
Two ways to transport CO2
1) Goes to bicarbonate dissolved in the plasma (most of it)
2) Gets put on to hemoglobin making carbamino hemoglobin
Pulse Oximeters
Measure heart rate and percent hemoglobin that is oxygenated. oxygenated and deoxygenated absorb light differently.
Functional Magnetic Resonance Imaging (fMRI)
Fully oxy hemoglobin is non-magnetic (diamagnetic)
Deoxy hemoglobin is magnetic (paramagnetic)
Glycosylated Hemoglobin (HbA1c)
Increase in glucose concentration results in irreversible glycosylated hemoglobin. This is seen in diabetic patients
Sickle Cell (Implications, AA Mutation, Malaria effect)
- deformed RBC
- tends to block capillaries and rupture them, causes bad bone development, gingival lesions, caries, perio disease, anemia, infections, renal failure
- GTG->GAG so Glu becomes Val (loses the charge)
- will accelerate destruction of RBC infected by parasites of malaria
- Tends to aggregate to large fibers
Isoelectric Point in Sickle Cell
Sickle cell is higher pI and when separating by electrophoresis it moves slower because it does not have the charge from Glu anymore
Thalassemia (Dental Effects)
imbalance of production of hemoglobin chains can be alpha or beta
Results in malocclusion, caries and gingivitis
Beta-Thalassemia
B chain of hemoglobin is not produced. So only an alpha chain is there resulting in insolubility and lack of cooperativity
Alpha-Thalassemia
Alpha chain is not produces enough so hemoglobin only has beta and binds oxygen with high affinity and no cooperativity
