Flashcards in M2M Unit one Deck (260):
What is entropy
Disorder or randomness in system
What is enthalpy?
Amount of energy (heat or bond energy) in a system
What is free energy?
The change in Gibbs free energy is the maximum work that a process can perform under constant pressure
What does a negative Gibbs free energy mean?
Reaction is favorable and spontaneous
What are high energy compounds?
Compounds that store chemical energy in their bonds which can be used for work on other compounds with relative ease
What is an oxidation-reduction reaction?
A reaction where a compound receives or gains electrons (reduced) while a compound that accepts electrons is oxidized.
First law of thermodynamics
Energy is neither created nor destroyed
Second law of thermodynamics
Entropy in the universe must always increase
What are the four different forms of energy?
Forms of potential energy?
1. Stored in bonds
2. Concentration gradients
3. Electric Fields from charge separation
4. Movement of charged particles down gradients of electrical potential
Formula for delta G?
= delta G0 + RT ln [products]/[reactants]
what is delta G0?
free energy change of a reaction under standard conditions (298 K, 1 atm, 1 M, pH=7)
formula for delta G0?
= -RTlnKeq = -2.3RTlog Keq = delta H - Tdelta S
formula for Keq
[C][D] / [A][B]
Negative H, Negative S = ?
spontaneous at low temperatures
Positive H, Negative S = ?
reaction is not spontaneous
Numerical conversion between free energy and redox potential?
delta G = nFdeltaE
E is the difference in reduction potential in volts
n is number of electrons transferred
What are the purines?
two ring nucleotide. Guanine and Adenine
What are the pyrimidines?
one ring nucleotide including Thymine and Cytosine
Ribose vs. Deoxyribose?
Ribose has a hydroxyl group at the 2' and 3' position on the sugar.
Deoxyribose only has a hydroxyl group on the 3' position of the sugar
includes the nitrogenous base(purine or pyrimidine) attached to 1' or sugar, sugar, and phosphate attached to the 5'
just the nitrogenous base and the sugar. Phosphate is excluded
Use Uracil instead of Thymine
order the solubility of the following: pyrimidine, purines, bases, nucleotides, nucleosides,
pyrimidine > purine; nucleotide > nucleoside > base
Medical Relevance of Gout and Lesch-Nyhan?
accumulation of purines which are insoluble in tissues. Causing joint pain
Disease(s) caused by accumulation of purines?
Gout and Lesch-Nyhan
Explain polarity of the phosphodiester bond
The phosphodiester linkage is between 3' OH group and the neighboring 5' phosphate group
Avery, McCloud, and McCarty contribution to science?
deduced that DNA is the genetic material through the pneumococcus experiment and killing mice
x ray diffraction suggested that DNA is a helical struture
Watson and Crick
DNA is a double helix and can semi conservatively replicate itself
Implications of Chargaff's Rules?
The ratio between G:C and A:T are approximately the same.
Molar ratios between purines (A+G) = pyrimidine (C+T)
The G:C And A:T ratio varies across different organisms
Describe Watson-Crick three dimensional model for DNA structure
DNA is a right handed double helix.
The sugar phosphate group is hydrophilic and on the outside of the molecule.
The bases are paired and stacked on the inside due to their hydrophobicity.
Major Groove of DNA vs Minor Groove of DNA
Major Groove is the gap between the curve of the same DNA molecule.
Minor groove is the gap between complementary DNA
What contributes to the stability of the double helix DNA in solution?
Magnesium ions in the cell neutralize the negative phosphate groups.
hydrogen bonding between base pairs
hydrophobic interaction of base stacking
Ways to further stabilize DNA?
increase salt concentration to neutralize phosphate negative charge
make DNA longer
increase G:C content
Methylation of Cytosine
DNA modification made by enzyme that methylates cytosine at the 5' position.
Needs a 3' G adjacent to it for methylation to occur
Nitrous acid or nitrosamine can be increased in a person that inhales cigarette smoke.
The nitrosamine can then cleave amine group in the DNA (deamination)
If cytosine has been (naturally) mythylated the deamination will make cytosine -- > thymine. Thus, there will be a T-G MISMATCH.
Can't be recognized as mismatch and 50% chance the mutation will remain
low ph can result in hydrolysis between sugar and the base
phosphate group is now prone to breakage
Can be repaired via DNA repair enzymes
UV cross linking
UV light causes covalent attachment of adjacent Thymines on the SAME strand.
Causes kink in DNA that disrupts replication
Mechanism of UV cross linking repair
nucleotide excision repair and TF2H
due to environmental exposure to coal, mustard gas (warfare), and cigarette smoke
nucleophilic reaction leading to alkyl or hydrocarbon being added to nitrogen
Not easily repaired and blocks DNA replication and transcription
Ways that nucleoside analogues can be used as drugs
1. Intercalating drugs insert themselves into DNA and alters DNA double helical structure. Interferes with DNA replication and transcription
2. Disrupts DNA synthesis (oldest target; today we focuson combination)
3. Inhibit topoisomerase from relaxing DNA which is necessary for DNA replication and transcription.
4. Covalently binding to base pairs
During DNA replication you have one old strand and one new strand
What does Bi-directional mean?
DNA replication occurs in two opposite directions starting at the origin site. ALWAYS 5' to 3'
small fragments of DNA assembled from the lagging template in the 5'-3' direction and then fused together by ligase
prokaryotes vs eukaryote origin sites
prokaryotes only have one origin for replication (they have less to replicate). Eukaryotes have multiple sites of oriin
Origin binding proteins
recognize the origin site. MCM in eukaryotes They recognize AT rich sequences
unwinds just ahead of the replication fork
Single strand binding protein
binds to each single strand of DNA and prevents dna from folding on itself or geting degraded
prevents extreme supercoiling of parental helix due to the unwinding at the replication fork
a topoisomerase found only prokaryotes and is inhibited by quinolones (antibiotic)
adds deoxyribonucleotides to the 3' hydrolxyl group of the RNA primer and adds dNTs in the 5' to 3' direction
DNA polymerase used in prokaryotes?
DNA polymerase I and III
DNA polymerase III
major replicative enzyme because it has a sliding clamp that keeps it attached = processive
Has 3' to 5' proofreading ability
DNA polymerase I
distributive and dissociates from DNA easily
It has 5' to 3' exonuclease activity to remove the RNA primer
replaces RNA primer with DNA in the 5' to 3' direction
It also has 3' to 5' proofreading ability
A DNA dependent RNA polymerase
needs DNA to add an RNA primer
joins fragments of DNA together
binds to the polymerase III to allow more processitivity. Keeps it tightly bound to DNA
an RNA dependent DNA polymerase that maintains chromosomal ends by making the telomeric repeat sequence from an RNA template
Diseases that are caused by mutations in genes that happen to mediate the nucleotide excision repair mechanism
What causes Thymine dimers
UV radiation causing linkage between adjacent thymine residues causing a bulge in the DNA helical structure
Consequences of unrepaired thymine dimer?
Disrupts replication because Pol III will fall of and bypass polymerase takes over leading to much more mutations
Best way to repair thymine dimer?
Nucleotide excision repair a much more versatile mechanism
Cause of bulky chemical adducts in DNA
chemotoxins that bind to DNA helix and disrupt the shape
Repair mechanism for chemical adducts?
Nucleotide excision repair
Cause of double strand breaks
double break of the phosphodiester back bone because of ionization radiation, oxidatitve damage, and spontaneous events
How to repair double strand breaks?
homologous recombination repair or non-homologous end joining
Homologous recombination vs non homologous joining?
Homologous recomibination requires extensive sequence homology between broken DNA and the DNA template. Very accurate
Non-homologous joining requires no sequence homology. Often inaccurate leading to deletions/insertions
How does uracil end up in DNA by mistake?
cytosine is deaminated and produces uracil in DNA now. Deamination is due to nitrous acid!
How to repair uracil mismatch in DNA?
base excision repair!
Consequences of having uracil in the dna?
problems with replicating, transcribing this part of the gene and recognition of transcription enzymes
Function of mismatch repair
fixes nucleotides that have been mistakenly added during DNA replication
Name the 3 types of excision repair
base excision repair
nucleotide excision repair
mismatch excision repair
Main difference between the three excision repair steps?
How the mistake is inItially RECOGNIZED
Recognition of a mismatched nucleotide by..
MSH and MLH
exonuclease vs endonuclease?
endonuclease cleaves the phosphodiester backbone of new strand of DNA
exonuclease CHEWS away the new DNA strand including the mismatch nucleotide.
Steps common to all three types of excision repair
1. Endonuclease-mediated cleaves the phosphodiester backbone flanking the damaged/mismatched nucleotide.
2. Exonuclease-chews DNA fragment containing the damaged/mismatched nucleotide.
3. DNA polymerase-mediated synthesis of the missing nucleotides by copying nucleotide sequence from the intact DNA strand.
4. DNA ligase-mediated sealing of the remaining nick in the phosphodiester backbone.
Hereditary non-polyposis colorectal cancer (HNPCC)
caused by mutation in the mismatch repair machinery
How can E.coli differentiate parental strand from daughter strand?
Daughter strand has not yet been methylated!
When is nucleotide excision repair used?
to remove damages that distort the DNA structure and block polymerase function.
Used to repair thimine dimers and bulky lesions and chemical adducts
Global Genome nucleotide excision repair (NER)
protein that recognizes damage anywhere in the genome
Transcription coupled NER
protein that only recognizes damage in transcribed regions
Disease caused by mutation in Global Genome NER
Disease caused by mutation in Transcription Coupled NER
When is based excision repair used
To repair DNA lesions that are missed by the NER process
these repairs don't necessary block polymerase function or distort DNA structure
Protein that recognizes Base excision repair
What is lesion bypass?
When cells have too much DNA damage for the "error proof" repair machinery to handle.
Cell uses DNA polymerase with loosened specificity to allow the continuation of replication.
What's unique about the bypass polymerase?
Lacks proofreading 3' to 5' and much more errors!
evolutionary conserved and is an example of "direct reversal" DNA repair.
it removes the methyl group from O6-methyguanine
MGMT is silenced via promoter methylation in 45% of glioblastomas
Cyclin dependent Kinase (CDK)
enzyme that is a protein kinase. The CDK subunit requires cyclin inorder to be activated
Retinoblastoma protein (Rb)
an inhibitor protein of the cell cycle aka a tumor supprossor
Need Rb inhibited in order for cell to divide and enter S phase
What protein(s) inhibit CDK?
CDKN (two types)
I. Cip/Kip Family
2.lnk4 family: p16,p15,p18, p19
What amino acids can be phosphorylated by CDK?
Serine, Threonine, and Tyrosine
What is mitogen?
proteins and peptides that eventually will cause the production of cyclinD1-3.
results in an increase in cyclinD1-3 proteins and more
CDK4/6-cyclin D1-3 active protein kinase complexes.
How is the cell size regulated?
In G1 phase, cell growth is coordinated with cell division at the “R” (restriction point). At the R point, the cell determines whether or not it is big enough to move on to S phase.
Ataxia Telangiectasia Mutated (ATM)
A serine/threonine protein kinase activated by DNA double strand breaks. Phosphorylates key proteins that initiate DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis
targets of ATM
p53, CHK2, and H2AX
Endogenous sources of double strand breaks
2. DNA replication single stranded DNA breask
3. Immune system rearrrangments
Exogenous sources of double strand breaks
1. Environmental radiation
2. Medical Radiation
Explain features of non-homolgous end joining
imperfect system wit ha loss of a few nucleotides occuring throughout cell cycle
What proteins work with non homologous end joining?
Ku recognizes the double strand break and recruits DNA-pKCs (DNA-dependent kinase). ATM triggers the DNA PKcs to autophosphorylate and recruit and phosphorylate artemis.
What does artemis protein do?
artemis is phosphorylated by DNA PKcs and is an endonuclease that cuts hairpins as well as 5' and 3' overhangs
Explain features unique to Homologous Recombination
requires a sister chromatid
only available during the G2 and S phase
When is homologous recombination used?
What can happen if you lose the ability to us non homologous joining and homologous joining?
Results in a loss of heterozygosity (loss of the entire gene and the surrounding chromosomal region)
tumor suppressor that regulates homologous recombination?
tumor suppressor that regulates non homologous recombination
What are the 3 classes of RNA in human cell
Examples of structural RNA
rRNA (RNA in ribosome)
snRNA (splicing and cell modification)
tRNA (moving RNA around)
Example of Regulatory RNA
miRNA and siRNA (downregulates gene expression)
Example of information containing RNA
Difference between RNA and DNA
RNA can spontaneously hydrolyze given the extra 2' OH group thus RNA can be cut up more frequently
RNA may have catalytic function like proteins
How does puromycin mimic amino-acyl tRNA?
strucuture is very similar to a tRNA carrying an amino acid. It binds to the tRNA acceptor region of the ribosome, transfers peptide, and terminate elongation making a defective protein
The RNA transcript is equivalent to which DNA strand?
The coding / nontemplate strand
E. Coli RNA polymerase does what?
transcribes all RNA in E. coli
eukaryotic RNA polymerase I
Makes ribosomal RNA (rRNA)
eukaryotic RNA polymerase II
makes messenger RNA (mRNA), small nuclear RNA (snRNA), and micro RNA
What is unique about RNA polymerase II
Has a C-terminal domain
eukaryotic RNA polymerase III
makes primarily tRNA
Which RNA polymerase is most abundant?
RNA polymerase I
How does alpha-amanitin block transcription?
found in death cap mushrooms and inhibits RNA polymerase II by binding to bridge helix and blocking translocation.
Non competitive inhibitor
How does rifampicin block transcription?
It binds to bacterial RNA polymerase and block RNA exit channel. Therefore no transcription can occur.
What is the sequence characteristic of a promoter site in humans?
Name four components of the RNA polymerase II pre-initiation complex.
Transcription factor TFII B,D,E,F, H
What's the significance of TFIIH?
It facilitates nucleotide excision repair. Think global NER (xeroderma pigmentosum) and transcribed NER (cockayne's syndrome).
Acts as a helicase to open up DNA
What is the significance of TFIID?
it is the TATA binding protein for RNA II polymerase!
Three major ways in which most pre-mRNA's are processed.
1. Capping: Adding 7-methyl Guanosine to the 5' end
2. Splicing: introns
3. Cleavage/Polyadenylation: 3' end
List the functions of the 5' cap of the mRNA.
2. 3' processing
3. nuclear export
4. Translation via Eukaryotic Initiation factor F4E
5' splice site for pre-mRNA
3' splice site for pre-mRNA
function of U1 in splicing.
recognizes 5' splice site
function of U2 snRNA's in splicing.
recognizes the branch point in pre-mRNA splicing between 5' and 3'
function of U2AF (protein)
binds to the 3' splice site AG
DNA Control elements
part of DNA sequence that act locally in terms for transcription
3. TATA Box / initiator
determines the site of transcription initiation and directs RNA polymerase II
What is Thalassemia?
Disease associated with mutation in DNA control elements
Leads to inherited anemia (low hemoglobin count) due to a mutation in the b-globin promoter region, resulting in lower production fo the b-globin protein.
What is Hemophilia B-leyden?
Disease associated with mutation in DNA control elements.
Mutation in Factor IX and associated with mutation in promoter region.
Affects males who don't make enough of Factor IX to help with blood clotting
What is fragile X syndrome?
Leads to mental retardation and atypical development of the face with enlarged testicles.
Expansion in the CGG count upstream of the FMR1 gene which results in excessive gene silencing.
What are examples of disease caused by mutations in DNA control elements?
Thalassemia, Hemophilia B-leyden, and Fragile X syndrome.
What are the two classes of transcriptional activators and repressors?
Sequence-Specific DNA binding proteins (6-8 bp) sequence
2. Co-factors that bind to the sequence specific DNA binding proteins
What are the different domains of transcriptional activators?
1. DNA binding domain - highly structured/highly conserved and binds to DNA.
2. Activation or repression domain: fairly unstructured/less conserved. Recruits other proteins
mutation in the homeodomain protein (helix, turn, helix) causes protein to bind more strongly and causes skull to close more prematurely
Androgen Insensitivity syndrome
Feminization or undermasculization caused by mutations in the zinc finger (two antiparallel beta turns, zinc, and alpha helix) . The androgen receptor no longer binds as well to the ligand binding domain and downregulates the transcription of genes controlled by male androgens.
Caused by mutation in the MITF gene = deafness and pigmentation defects
Diseases caused by mutation in the DNA sequence specific domain binding region
Craniosynostosis, Androgen insensitivity, and Waardenburg syndrome.
Describe combinatorial control as a mechanism for controlling gene expression
The Zinc finger for example can form heterodimers. If each monomer of the heterodimer has a different DNA binding specificity, the formation of heterodimers will increase the number of potential sequences to which that family of sequence specific transcription factors can bind
repeating unit of chromatin that has 147 bp of genomic DNA wrapped around an octamer of histone proteins
What is the role of the Swi/Snf protein?
Uses energy of ATP hydrolysis to break histone-DNA and expose the parts of DNA needed. Slides the histone octomer along the DNA
What are Histone Acetyl Transferases (HATS)
unwinds DNA to expose areas for transcription. Changes outer histone proteins from lysine --> acetyl (positive to negative) making DNA less stable
Histone Acetylations Diseases
Leukemia and Rbuinstein-Taybi Syndrome
acts as an antagonist to estrogen, binding to the estrogen receptors as a ligand without providing dimerization-- thus effectively preventing estrogen from binding to its site of action and preventing the transcriptional effect of estrogen receptors.
Example of how nuclear hormone receptors is controlled
estrogen crosses the membrane and binds to estrogen receptor. ER has a DNA binding domain and zinc finger binding motif that recruits other proteins
example of a sequence specific DNA binding protein regulated by nuclear entry
NF-κB: normally bound to IκB hiding the Nucleus signal (NLS) Under certain conditions, IκB is phosphorylated, which targets it for degradation. Degrading IκB shows the NLS of NF-κB to migrate into the nucleus and affect transcription for inflammation response
How is β-catenin regulated?
APC targets beta-catenin (a cell proliferation activator protein) when APC triggers beta catenin for protease degradation. If there is an APC mutation there is a huge build up of beta-catenin which can now enter the nuclease and cause cell proliferation
Most common cause of colon polyps?
caused by mutations in the APC gene, resulting in insufficient degradation of (and thus proliferation of) B-catenin.
What is the function of ID proteins?
inhibitor of the E box proteins and regular helix loop helix protein
Steps to 5' capping
1. Triphosphatase (removes phosphate)
3. Guanine 7 methyl transferase
What is the start codon?
What are the stop codons
UAA, UAG, UGA
There are multiple codons for a single amino acid
What type of DNA repair mutation causes hereditary non-polypsosis colorectal cancer called Lynch syndrome?
Functions in transcription and DNA repair (nucleotide excision repair)
part of the TFIIH protein
part of the TFIIH protein that phosphorylates the C terminal domain on RNA polyermase II and triggers promoter clearance
C terminal domain
unique feature of RNA polymerase II that gets phosphorylated by CDK 7 (part of the TFIIH protein) and triggers promoter clearance
single stranded binding protein for e. coli that keeps the DNA from getting cut up during replciation
is the eukaryotic SSB protein
the sliding clamp for eukaryotes
What's the eukaryotic equivalent to DNA polymerase III
DNA polymerase gamma and epsilon
What's the eukaryotic equivalent to DNA polymerase I
DNA polymerase alpha
caused by mutation that disrupts the splicing of the fibrillin gene (fibrillin is a connective tissue important for integrity of blood vessels)
is a cell surface glycoprotein that determines migration of cells. It therefore contributes to tumor metastasis and can e used for diagnostic purposes
3 types of high energy phosphate bonds?
1. Phosphoanhydride (ATP)
2. Phosphocreatine (P-N)
3. Phosphoenolpyruvate (C-O-P)
What is the role of excision endonuclease in nucleotide-excision repair of DNA?
They cleave the damaged DNA strand at either side of a lesion.
haploinsufficiency of CREB binding protein (CBP) which is a HAT involved in the activation of many genes. It is a multisystem disorder.
Shine Delgarno Sequence
Purine rich sequence (AG) that bacterial ribosomes use to indicate the start site for translation. It's upstream from the AUG codon
How does the small subunit (30s) of the ribosome bind to the mRNA
using the Shine Delgarno Sequence
First set of initiation factors for bacterial translation to occur
Recruitment of IF1 and IF3 proteins bind to the 30s subunit
Now mRNA cand bind to the subunit via it's shine delgarno sequence
AUG start codon now in the 30S subunit P site
Second set of initiation factor(s) for bacterial translation to occur
IF2 helps deliver a special initiator formylmethionine tRNA which attaches to the AUG start codon
Last step in the initiation process of translation in bacteria
GTP hydrolysis on IF2 leads to the release of all other initiation factors.
Binding of 50S subunit can now occur
What type of energy is used to combine the 50S subunit and 30S subunit for bacteria ribosomes?
What is the Kozak sequence
A sequence that occurs on Eukaryotic mRNA (gccRccAUGG) recognized by ribosome as a start site.
different start codons have different “strengths” depending
on their Kozak context.
Applies to prokaryotic mRNA. One transcript alone can encode for many different proteins
Aminoacyl tRNA synthetase
An enzyme responsible for adding the correct amino acid to the tRNA with the correct anticodon by hydrolysis of ATP.
peptidyl transferase center (PTC)
resides in the ribosomal large subunit and catalyzes the peptide bond formation using ATP as energy
What triggers movement of the mRNA and tRNAs exactly one codon in the 3’ direction?
Elongation Factor 2 (EF2) and GTP hydrolysis
How much energy is required to make a peptide bond?
4 high energy bonds
2 ATPs to charge TRNA
1 GTP to deliver AA to TRNA Asite (via EF1)
1 GTP to translocate one codon in the 3' direction
What terminates translation?
This is a PROTEIN driven event. No TRNA can recognize the stop codon and a release factor comes in and recognizes the stop codon.
GTP hydrolysis causes cleavage
the first elongation factor in Eukaryotes that brings tRNA to the A site
bacterial elongation factor equivalent to EF1A
The codon is changed so now is encodes a different amino acid.
Mutation that leads to a premature stop codon
Mutation that leads to the removal of a stop codon (opposite of sense)
internal ribosome entry sites (IRES)
cap independent process in eukaryotes that viruses can take advantage of after shutting down the host's cell cap dependent process
4E-binding proteins (4E-BPs)
sequester and bind to eukaryotic initiation factors 4E (EIF4e) and block it from recognizes the 5' Cap
Result from phosphorylating 4E-BPs?
These 4E binding proteins can no longer bind to eukaryotic initiation factor 4E. Therefore, EIF4e can recognize 5' cap for translation to occur
Inhibits m-TOR which normally phosphorylates 4E binding proteins so that EIF4E can bind to 5' cap and initiate translation.
This drug inhibits cell proliferation
master controller of cellular process that phosphorylates 4Ebinding proteins so they cannot block the function of EIF4E
Eukaryotic initiation factor 2 alpha (eIF2-alpha)
important for bringing in the initiaor tRNA which attaches to the AUG start codon in the ribosome
What's the outcome of phosphorylating eIF2-alpha
eIF1-alpha activity is inhibited and transcription is stopped.
Interferon and eIF2-alpha
An interferon indicates cell has been infected with virus and triggers phosphorylation of eIF2 to stop viral proteins from being made.
Disorder caused by frameshift mutation
Disorder caused by a sense mutation where stop codon is removed
An example of how mRNA can be altered after its been made
protein production in the intestines is shorter than in the liver
transport transferrin/iron into the cell
Sequesters excess iron
Iron response element
RNA stem loop structure found in mRNA that can bind to iron response binding protein (when low concentration of iron)
Iron response binding protein
binds to iron and regulates expression of ferritin and transferrin receptor
Hydrophobic amino acids
Aromatic amino acids
Try Tripping with Phenylalanine
Polar uncharged amino acids
Great Asshole, Please Come Take Shrooms
Polar Positively Charged
Polar negatively charges
Good ' Ayyy
AA is protonated
AA is deprotonated
Essential Amino Acids
Private Tim Hall = PVT TIM HALL
Break down of collagen which needs hydroxyl group on proline for strength. Vitamin C delivers the hydroxyl group
Carboxylation of Glutamate
Important for blood clotting and carried out by Vitamin K
Deficiency in Vitamin K
improper blood clotting
prevents carboxylation of glutamate and acts as anticoagulant
Congenital Disorder of Glycosylation (CDG)
glycosylation cannot occur on asparagine and impact hydrophilicity in secreted and cell surface proteins
An bcr-abl protein kinase inhibitor that binds to the bcr-abl kinase domain so it can no longer bind to its substrate and cause uncontrolled blood cell proliferation
Add ubiquitine to mark cell destined for degradation by proteasome
Peptide bond characteristic
Has partial double bond and therefore no rotation.
formed from dehydration reaction of COO- and NH3+
Bond from alpha carbon to carbon of peptide bond/carbonyl (C2-C1)
Bond from amide nitrogen to alpha carbon (N-C3)
Strongest non covalent bond
Strong helix formers
Alanine and Leucine
Proline and Glycine
Introduction of proline in the alpha helix causes breaks and destabilization of hemoglobin
Structural fibrous proteins mostly made up of alpha helixes
Globular proteins made up of alpha helices
Structural fibrous proteins mostly made up of beta sheets
Fibroin - silk, spider webs
Globular proteins made up of beta sheets
Beta turn AA
Dont like to be in Alpha or Beta
Proline and Glycine
Amino Acids that prefer Beta conformation
Hydrogen bond between 1st and 4th amino acid
Can be in trans or cis (rarely) conformation
AA acids most frequently used in loops and turns
Proline and Glycine
multiple stable globular units that have independent functions
Formed by multiple polypeptides into a larger functional cluster
Each chain has Glycine nad Proline left-handed helix
Together makes a right-handed superhelical triple helix
Explain how to use Kd to represent binding strength.
Kd is the dissociation constant
Kd= [ligand] when 50% of ligands are bound
Main oxygen storage in mammals. Stores heme in the center of protein so no risk of oxidation
first binding event increases affinity at remaining sites
no oxygen is bound to hemoglobin / low affinity for oxygen
relaxed state induces conformational change and has high affinity for oxygen now
Oxygen binds well at higher pH (like in the lungs)
Oxygen is release well at lower pH (tissue)
Ribonuclease refolding experiment
Used reducing agent to denature Ribonuclease A, then after dialyzing out the urea it slowly refolded and restored almost 100% of activity
All the information needed to fold the protein correctly is embedded in the primary amino acid sequence
The environment provided by the inside of the cell is not always required in order for proteins to fold correctly
The protein does not explore all possible structures while folding, there is instead a pathway it follows. (Levinthal’s paradox)
proteins don’t sample all confirmation to achieve folding state
it would take way too long!
Heat Shock Proteins (Hsp70)
induced at elevated temperatures and binds to hydrophobic region of unfolded proteins to prevent aggregation, can also help transport some proteins across membranes in unfolded states, works with other heat shock proteins
consists of a cap and two 7-subunit rings. The hydrophobic region of the unfolded protein binds to the hydrophobic region of the chaperonin then with some ATP and a conformational change of the chaperonin the protein is folded at least partially so that it can only continue to the final native shape.
Ex: GroEL/GroES complex in E. coli.
protein disulfide isomerase (PDI)
reduces improper disulfide bonds and reform them correctly
Peptide prolyl isomerases (PPI)
Speeds up process of trans to cis confirmation in proline
Beta-synuclein misfolds into Lewy Bodies (protein agreggrates)
Generalized protein misfolding in the rest of the body leading to a variety of disease (from Type II diabetes to Cardiac amyloidosis)
Identify the secondary structure changes in prion disease
alpha -> beta sheets
gel filtration chromatography
separates proteins by size