Test 2 Chapters 6,7 Flashcards
What does “salting in” do?
Increase in solubility of a protein with increasingly low salt concentration.
What is salting out
The decrease in solubility of a protein with increasing high salt concentrations.
When salting out what does adjusting pH to match pI result in?
Makes the protein less soluble b/c no net charge.
What is gel electrophoresis? And what is it used for?
Migration of ions in electric field. Uses: Identification: if band matches a known band. Purify Quantity: use a molecular weight loader
Types of gel electrophoresis and their uses:
- PAGE: Separates by size, shape, and charge.
- SDS-PAGE: Separates by charge alone.
- 2D gel electrophoresis: protein settles at pH corresponding to pI
What do the proteins travel toward in PAGE? And what is the pH?
Toward the anode (+); pH=9
What is PAGE electrophoresis also known as? And why
Native gel, the protein is left in native conformation and is not denatured.
What does the SDS in SDS-PAGE stand for?
And what is it and what does it do?
Sodium dodecosulfate: [CH3-(CH2)10-CH2-SO3] Na
It is an amphophilic detergent, that forms micelles with negatively charged molecules on the external surface, and denatures proteins by hydrophobic interaction.
When running SDS-PAGE, what are common chemicals to reduce disulfide bonds aiding to unwind the protein
2-mercaptoethanol (BME)
Dithiothriotol (DTT)
How does 2D get electrophoresis work.
Through a combination of SDS-PAGE and IEF (Isoelectric focusing). This gives each protein a unique position based on M.W. and pI.
The protein will settle at the pH corresponding to pI
Reasons for protein sequencing
Structure/function relationships Diagnostic tests: >(therapies for mutations) (ID them) >Evolutionary relationships >>sequence comparison
Steps of protein sequencing.
- Separate the sub-unites
- Cleave polypeptides
- Edman Degredation
- ID via chromatography
- -repeat-
- Determine sequence
- Repeat w/o reducing agent to ID cystine
- Add to Database
If you want to alkylate a thiol group what is used, and why?
Iodoacetate; prevents the reformation of disulfide bonds.
What type of molecule is used to cleave peptide bonds.
Endopeptidase (protease)
When cleaving peptide bonds what does trypsin?
Cleaves peptide bond between arginine and lysine, as long as proline does not follow.
Cleaves scissile bond.
Example of chemical cleavage?
Cyanogen bromide
What does cyanogen bromide do?
C-side of Met
What is the process for removing the N-terminus amino acid one at a time.
- Phenylisothiocyanate (PITC); AKA “Edmund’s reagent”
»reacts to form (PTC) adduct on N-terminus - Trifluoroacetic acid: cleaves the N-terminus.
What are the three common databases?
ExPASy
PIR
Uniprot
What does mass spectrometry measure?
Mass spectrometry accurately measures the mass-to-charge (m/z) ratio for ions in the gas phase.
M= ion’s mass
Z = its charge
What problem did electrospray ionization fix?
Allows for the protein to remain intact.
Intact charged macromolecules: (lysine, histamine, arginine, N-terminus (per subunit).
What conditions are ESI normally run under?
And
How are the samples separated?
Acidic and by weight and charge
What is a tandem mass spectrometer and what does it do?
It is two mass spectrometers in a coupled series.
First the polypeptide is fragmented into short chains.
Fast process and can measure post-translational modifications, but is unable to distinguish:
Ile from Leu and Gln from Cys (0.036 Da difference)
Where is cytochrome C (Cyt C) found and what does it do?
In mitochondria, and carries electrons from complex 3 to 4.
If a specific residue is found in the same spot in all samples what can be infered?
Example: 73 lysine
This means it is vital for function since if it was changed it may no longer function.
What does it mean if there is a lot of variability at a certain place in the code:
Example
At 89 there are constant changes.
This indicates it is not as vital since the variability would other ways cease it from functioning.
How does protein evolution occur, and what are the types?
A change in genes which can change the primary structure of a protein.
Types:
>Beneficial or Neutral: rare and due to natural selection or genetic drift.
>Deletions: by far the most common.
How do proteins reveal evolutionary relationship?
Sequence alignment, and in Cyt C: conserved residues.
What are homologous proteins?
Evolutionarily related proteins
vs % sequence identity
How similar two proteins are without being related.
Invariant residue definition?
Conserved, usually essential for function
Conservatively substituted residue definition.
Change residue to one with similar properties.
Asp—->Glu
Ser—–>Thr
Hypervariable residues definition
A.a. not specific to function
|»_space;may be a glorified “space holder”
How does most protein evolution occur?
Gene duplication and gene segment duplication.
What are the differences between adult hemoglobin and fetal hemoglobin?
Adult: a2B2
Fetal: a2y2
Fetal has higher affinity for oxygen.
What does paralogous mean?
Two genes with different functions that evolved during gene duplication.
Pseudogene?
Gene with no new function and doesn’t retain the original function.
What does Othologous mean?
Homologous proteins generated by speciation, that often have the same function.
Rate of sequence divergence
Rate of evolution varies among proteins.
>b/c rate of successful mutations vary
>relatively lower for a particular protein.
Rate is dependent on a.a. relevance to function.
>structural stability.
What is a domain?
Evolutionarily conserved structures (40-200 a.a.)
Usually have structural similarities.
What is domain shuffling and related concepts?
Gene segments that encode for protein domains are copied and inserted into other positions in a genome to generate new genes that encode proteins.
Many proteins made up of multiple domains,
Evolution is faster with domain shuffling.
Defining structures of primary structure?
A.a. sequence and disulfide bonds.
Defining structures of secondary structure?
Spatial arrangement of back bone with minor considerations of side chains.
> a helix
> B sheets
Defining structures of tertiary structure?
Spatial arrangement considering side chains; determines where hydrophobic and hydrophilic subunits are.
Defining structures of quartenary structure?
Includes subunits of proteins that multiple.
What is the nature of the peptide bonds.
Rigid planar structure.
About 10% of Proline are?
CIS
Backbone C main chain can be described by torsional angles.
Cα----N = Φ Cα----C = Ψ
What do the different colors indicate on the Ramachandran Diagram.
Blue = sterically allowed Φ and ψ angles for all residues except Gly and Pro.
Green = indicate the more crowded (outer limit) angles.
Yellow = represent conformational angles of several secondary structures.
Two exceptions to the Ramachandran diagram?
Proline: Φ angles limited to around -60
Glycine not sterically hindered.
What are the two common secondary structure, structures?
α - helix
Β - sheets
Features of an α-helix?
Right handed turn
3.6 residues per turn
5.4 A pitch
Hydrogen bonding between C=O (nth residue) and N-H (n + 4 residue)
What are the defining features of B-sheets?
H-binding with neighboring strand Pleated sheets 2 main types: >parallel (has looping effect) >anti-parallel R-groups within a strand alternate sides Slight right hand twist, for each strand.
What are the features of a reverse turn?
180 degree flip back bone Usually found on protein surface > H-bonding 4 a.a. > Two types >> Type 1 >> Type 2 - contains proline
Keratin is an example of what type of protein?
Fibrous
What are the two types of keratin and what are they found in?
a - keratin: Hair, nails, horns, hooves, skin
B - keratin: scales, feathers, beaks
What are the features of a - keratin?
2 a-helices in a left handed coil (coiled coil) (super helix) 7 residue pseudo-repeat 1 and 4 are usually non-polar >>roughly same as a turn >>results in a hydrophobic strip.
From keratin to hair.
- Two keratin polypeptides form a dimeric coild coil.
- Two staggered rows of head to tail associated coiled coils form a protofilament.
- The protofilaments dimerize to form a protofibril, four of which form a microfibril.
Keratin has many _____ residues.
The number of which dictates if it is _____ or _____.
Cysteine; soft and hard.
What is the most abundant vertebrate protein?
Collagen
Where is collage found?
Skin, bone, teeth, blood vessels, cartilage, and tendons.
What is the structure of collage?
Triple helix, composed of 3 L-handed helices wrapped in a right handed twist. >>Composed of: >>> ~33% Gly >>> 15-30% hydroxylated proline >>> Some hydroxylated lysine
How do collagen fibers crosslink?
Via covalently bonded side chains, and joint stiffness.
What was the third type of secondary structure?
Non-repeating “coils”, still order but irregular.
>not a random coil since that would denote no secondary structure.
What are the features of a tertiary structure, and how can they be visualized?
Folded secondary structure, each atom has an address.
Molecular graphics program can be used to visualize it.
Equation for fractional saturation of oxygen of Mb
Yo2 = (pO2)/(KD)+(pO2)
When pO2 = KD, Yo2 =0.5
What is the p50 for Hb?
Hb p50 = 26 torr
What is the p50 for Mb
Mb p50 = 2.8
What is the Bohr effect?
As you go down in pH the Hb has less affinity for oxygen.
Effects:
Enhances oxygen transfer
T–>R causes some pKs to decrease (His & Lys)
Explain the cross link structure of collagen.
Gly-X-Y
>X is often proline
»this prevents alpha helix formation
>Y is often Hyp, and sometimes Hyl.
> Glycine fits easily in the center because of its little steric hinderence.
>Staggard so that cross section results in all 3
What is x-ray crystallography?
fun x-ray beam and diffraction pattern.
What are the 4 structural motifs?
- BaB
- B hairpin
- Greek key
- alpha-alpha
- Omega loop
- Zinc Finger
Side chain locations:
Often found in the internal hydrophobic region of the protein
non-polar a.a.
Side chain locations:
Mostly found on the polar surface of the protein
Charged groups, except the backbone
Turns and loops
Side chain locations:
Where are the uncharged polar side chains?
Both internal and external, this decreases overall polarity of the group this is the backbone.
Side chain locations:
What might you find at a catalytic site?
Histidine
Side chain locations:
Structure of trans-membrane proteins?
Hydrophobic patch that allows two hydrophobic areas to interest.
a, b, a/b protein classification:
Cyt b ?
and motif?
a
motif: aa
a, b, a/b protein classification:
Immunoglobulin fragment?
and motif?
B
motif: Greek key and B hairpin
a, b, a/b protein classification:
Lactate dehydrogenase
a/B
motif: BaB
Protein topology:
What is a B-barrel composed of?
B hairpins
Protein topology:
What is a jelly role barrel composed of?
Greek keys
Protein topology:
What is a aB-barrel composed of?
BaB
Quaternary structure:
Homodimer?
same subunits
Quaternary structure:
Heterodimer?
different subunits
Quaternary structure:
Protomer
Identical units of oligomers (few to 20 a.a.)
aB is a protomer
Quaternary structure:
What is a dimer of two protomers?
Diprotomer
Quaternary structure:
What interactions occur between subunits?
Hydrophobic, hydrogen bonds, interchain disulfide bonds.
What is a chaotropic agent?Example of a chaotropic agent?
Increase solubility of non-polar groups.
Guandidium, urea
Mechanism of protein folding?
- Begins folding as translated.
- Secondary structure forms due to hydrophobic collapse, and forming molten globule (beginning tertiary structure).
- Domains fold individually
- Then sub units associate.
How are wrong disulfide bonds corrected?
Using the protein: Disulfide isomerase
This corrects non-native disulfide bonds by breaking and reforming the correct bonds.
Concept of molecular chaperones?
Isolate folding proteins. (in vivo high protein concentration)
Example of chaperones?
Heat shock proteins
What heat shock proteins were discussed in class?
HSP 70 (DNA-K) >Cotranslational or immediately after translation Gro EL/ES chaperonin >induce conformational changes HSP 90 cell signaling
Inherited protein misfolding?
Amyloidoses
Four prion diseases?
Mad cow–>BSE
Kuru
CJD
Scrappie
Mb structure
8 a helices globular Heme group Porphyrin derivative (4 pyroles) Fe (II) coordinated by 5 nitrogens and held in place by Val and Phe
What role does the E7 histidine play?
Prevents heme oxidation Fe(II) to Fe(III)
color of deoxy Mb
Purple
Color of oxy Mb
Scarlet
