Proteins Exam 1 Flashcards
silent mutation
no change in AA sequence
non-sense mutation
causes truncated protein by creation of stop codon
missense mutation
causes a change in AA sequence
frameshift mutation
causes all AA after frameshift to be mutated
insertion
deletion
Fred Sanger
1918-2013
inventor of sanger protein sequencing method
N - alpha C
phi bond 111degrees
alpha C - C bond
psi bond 120 degrees
Edmans Sequencing
Slow with multiple steps
Need pmol minimum of protein
Huge problem with background noise from reactants incorrectly bound
Only modification that can be detected is disulfide bonds
Cannot determine blocked (on N-terminus) or post-translationally modified amino acids
Properties of Mass Spectrometry
Fast
fmol of protein
Amenable to High-Throughput
Blocked/Modified peptides
Linus Pauling
1901-1994
Proposed alpha helix and beta sheet structures
alpha helix
3.6 AA per turn
1 – 10 loops
Side chains point out
Stabilized primarily by
Hydrophobic Interactions
N of an amino acid forming H-bonds with C of carboxyl group 4 AA ahead of the first AA
Electromagnetic interactions
Polarity of a-helix
what bonds stabilize alpha helices
Hydrophobic Interactions
Entropy because phi and psi angles fixed ~60°
H-Bonds
Vander Waal forces
Salt bridges
Helix dipole
what is the secondary structure loop
Interconnecting and non-structured elements connecting structured secondary elements
what do beta turns usually contain
proline and glycine
who developed X-ray crystallography techniques for polypeptide structure determination
max perutz and john kendrew
who developed NMR based techniques for polypeptide structure
richard ernst, Kurt Wuthrich and Albert Overhauser
what is a domain
Subsection of a polypeptide
Usually linked by a unstructured region of the polypeptide
Often contain separate functional abilities
what is tertiary structure controlled by
Constraints by type and positioning of secondary structure
Interactions between amino acid residues
what are the stabilizing factors in tertiary structure
Hydrophobic Interactions
Electrostatic Interactions
Covalent Linkages
Disulfide bonds
tertiary structure motif
A tertiary structural and/or functional sequence element found in multiple polypeptides of diverse origin that consist of similar secondary structures arranged in similar spatial organization that may play similar functional roles.
tertiary structure motif examples
helix-loop-helix
four helix bundle
zinc finger
greek key topology
hairpin structure
beta sandwich
beta barrel
what do beta barrels form
pores and channels
family definition
Polypeptides that have high levels of Sequence, Structural, and/or Functional similarities. Traditionally, evolutionarily related polypeptides. Now may refer to polypeptides whose structure and/or function only are similar but are not evolutionarily related.
superfamily definition
Polypeptide families that have Sequence, Structural, and/or Functional similarities (less than individual polypeptides at the family level). Traditionally, evolutionarily related polypeptide families. Now may refer to those whose structure and/or function are similar but are not evolutionarily related.
Van’t Hoff Analysis
Melting Curve (transition curve) and Van’t Hoff’s analysis allow determination of the thermodynamics of protein stability
Things that can be determined
Fraction of protein natured vs. denatured.
Keq of protein(s) at a given temperature
ΔH
ΔS
ΔG
E= the sum of (E NOE + E bond + E VWF + E angle)
Van’t Hoff Analysis Assumptions
Assumptions:
DH and DS are not temperature dependent
Limitation because Keqcan be measured only over a narrow range of temperatures
Post-Translational modifications
phosphorylation and sumoylation
glycosylation
methylation
acetylation
acylation
amidation
sulfation
proteolytic Cleavage
glycosylation
attachment of sugars
where does glycosylation happen
extracellularly
two types of glycosylation
n-linked and o-linked (S,T)
functions of glycosylation
Guide protein folding/assembly
Targeting and Trafficking of Proteins
Aid Ligand binding to receptors and elucicating the biological response
Stabilize Protein
Regulate ½-life of proteins (sialic cap on N-Terminus)
methylation
addition of a methyl group, mono, di, or tri
on which amino acids does methylation occur
R or K
function of methylation
Linked with protein function, but exact action is often not predictable.
Effects must be empirically determined.
Mono-, di-, or tri-methylation can have opposite, combined or opposing effects.
acetylation
N-acetyltransferases transfer acetyl group from acetyl CoA
where does Acetylation occur
on the N terminus of some proteins, can occur co-translationally or post-translationally
What structure is added in acylation
addition of lipids to proteins
where is acylation found?
Found in all eurkaryotes and in viruses
Found on a variety of proteins including structural, cytoplasmic and membrane proteins
most common lipids attached in acylation
Palmitic acid: 16-C saturated fatty acid
Ester or thioeser linkage to S, T or C
Post-translationally added
Complex Regulation
Myristic acid: 14-C saturated fatty acid
Linked to N-terminus of G
Co-translational Modification by myristoyl CoA:N-myristoyl transferase
function of acylation
Function is not well-defined:
Not likely interaction with membranes
Maybe protein complex stabilization or protein:protein interactions
amidation
amide group attached to C-terminus
where is amidation found
on short peptides
amidation function
Function not well understood
May help binding of regulatory peptides to certain receptors
May stabilize the structure and functions of proteins
sulfation
added sulfate groups mediated by sulfotransferases
where does sulfation occur
higher eukaryotes
YST
function of sulfation
protein:protein interaction
proteolytic Cleavage
cleavage of protein after translation
function of proteolytic cleavage
Create Functional mature protein from “pre”-protein
EPO
Target protein to organelle or extracellularly
what to consider for protein purification
level of protein expression
source of protein
location of protein
fusion tag
physiochemical properties
purpose of purification
physiochemical properties
size
solubility
charge
hydrophobicity
Function of immunoassays
Usually just measure protein presence, but not activity
why are immunoassays good
High Affinity molecules
High Specificity
High sensitivity
Short assay times
Easily automated
Recombinant proteins with Fusion Tags
bioassays
know protein is present and active
pros and cons of bio assays
pros: examine function of protein
cons: costly, imprecise
which is preferred bioassays or immunoassays
bioassays
plant and fungus cell disruption
Waring Blender
Glass Beads with freeze/thaw
Animal cell disruption
Potter homogenizer
Dounce homogenizer
Osmotic Shock
Freeze/Thaw
Waring Blender (for tough tissues)
microbial cell disruption
Chemical methods
Lysozyme
Detergents, ionic
Organic Solvents
Antibiotics
Alkaline Conditions
Chaotropic Agents
Physical Methods
Sonication
Glass beads
Microbial Homogenization
cheapest way to get rid of whole cells and cell debris
filtration
0.2 micrometer pore sizes removes all bacteria
materials of filters
cellulose acetate or cellulose citrate
nylon
PTFE
PEG
polyethylene glycol
affinity partitioning
uses a ligand of high specificity to protein
diafiltration
Same as ultrafiltration except reservoir of filtrate kept constant
Often pellet continuously resuspended and recycled through filter.
chromatography
separation of proteins based on differential partitioning between a stationary phase and a mobile phase
chromatography steps
Equilibrate column to correct conditions
Load sample onto column
Irrigate column
Elute proteins by changing properties of mobile phase
Monitor Proteins in each fraction by absorbance at 280 nm
Assay fractions with protein for protein of interest
chromatography size exclusion chromatography
Also called “gel permeation” chromatography
Matrix is porous, not adsorptive
Large proteins run fast, small proteins slow
As do irregular shaped proteins
Difference is that small proteins can enter more matrix pores
chromatography size exclusion column composition
Usually made of cross-linked polymer fibers of molecules like dextran, agarose, acrylamide, and vinyl polymers
con of size exclusion chromatography
dilutes protein
pro of ion exchange chromatography
Most popular and most employed in at least one step of protein purification
Easy to use
Very high resolution of proteins
Concentrates proteins
Scalable for industry
ion exchange chromatography
Based on reversible charge of protein at different pHs of mobile phase
pH values above the pI are more negative, pH values below the pI are more positive
anionic and cationic
anionic ion exchange chromatography
Anionic
Bind negatively charged proteins
Groups attached to matrix are positively charged
Aminoethyl
Diethylaminoethyl
cationic ion exchange chromatography
Cationic
Bind positively charged proteins
Groups attached to matrix are negatively charged
Sulfo
Carboxymethyl
reverse phase chromatography
organic solvent to elute
hydrophobic interaction chromatography
Patches of hydrophobic residues on protein surface
Can exploit these patches of hydrophobicity to purify protein
Uses hydrophobic groups
what resins and groups are used in hydrophobic interaction chromatography
Resins
Agarose
Sepharose
Groups
Octyl-
Phenyl-
two ways to elute via hydrophobic interaction chromatography
Eluted by increasing ionic strength of solution
NaCl or ammonium sulfate
Attracts water away from protein
Eluted by Lowering polarity of buffer
EtOH
Ethylene glycol
hydroxyapetite chromatography
Natural mineral in rock and bones
Mechanism of binding not well understood
Elution by irrigation with potassium phosphate buffer
Used as last resort
most powerful kind of chromatography
affinity
affinity chromatography
Most powerful because of its specificity and selectivity
Elution buffer changes disrupt bonds between ligand and protein
pH
Ionic strength
Agents reducing solution polarity
Detergents
Ethylene glycol
Competing ligands
Combination of the above
limitations of affinity chromatography
Reagents expensive
Reagents often unstable
Techniques to couple ligand are complex, hazardous and time-consuming
Ligands leaching from column
Reduces capacity of system
Noxious products end up in the eluant
immunoaffinity chromatography
Antibodies covalently attached to matrix
Very high specificity
Sometime difficult to desorb
Often changes buffer pH to desorb
Glycine-HCl at pH 2.2 – 2.8
lectin affinity chromatography
Lectins from plants and some invertebrates
Binds glycoproteins
Examples of lectins
Concanavalin A
Soybean lectin (SBL)
Wheat germ agglutinin (WGA)
Bind at close to neutral pHs
Desorption occurs by changes in buffer or adding free sugar molecules
dye affinity chromatography
Blue Dextran Dye in gel filtration columns (agarose)
Triazine Dye (Cibacron Blue F3G-A)
Dye has affinity for proteins
advantages of dye affinity chromatography
Dye available in bulk
Coupling is easy and non-toxic
Dye resistant to chemical, physical and enzymatic degradation
Protein binding capacity high
Easy to elute proteins
disadvantages of dye affinity chromatography
Mechanism not well understood
Has aromatic groups
Has sulfonate groups (negative charge)
Makes hydrogen bonds
immobilized metal ion affinity chromatography
Pseudo-affinity purification
Exploits weak bonds between metals and basic residues on protein surface
Fe2+
Ni2+
Cu2+
Co2+
Charged with metal
Binds basic groups – usually H
Elution solution uses
lower pH buffer to deprotonate the ion
Chelating agent – eg, EDTA
Used prominently for purification of recombinant proteins
chromatofocusing
Using a column and mobile phase of two different pHs
Forms a continuous pH gradient along column
Both column matrix and mobile phase buffer need a good buffering capacity
Sample applied in running buffer (mobile phase) whose pH is below that of the column
Negatively charged proteins adsorb into column
Positively charged protein go to part of column where column pH = their pI
But pH of column is constant getting more basic
Thus proteins move sequentially down the column based on the pI from most acidic to most basic
Elute in this order as well
High degree of protein resolution
HPLC
Other Chromatography is low-pressure
Higher pressures give
Quicker runs
Sharper peaks
Used at analytical or preparative scale
higher elution rates and better elution peaks
types of columns in HPLC
made of high-grade steel
Ion exchange
Gel filtration
Hydrophobic interactions
what kind of proteins is HPLC good for
small and extracellular proteins
advantages of HPLC
Superior resolution
Decreased fractionation times
binding tags on proteins
His tags and IMAC
Glutathione-S-transferase and Glutathione
N-terminus or C-terminus
Can also be used as antigenic epitope
steps to purify recombinant proteins
Engineer cells
Equilibrate IMAC column
Grow cells
Add IPTG (Isopropyl b-D-1-thiogalactopyranoside)
Lyse Cells
Apply lysate to column
Incubate to bind protein
Wash column at least 2 times
Eluted with Imidazole
or by lowering pH of elution buffer
why is it necessary to remove a tag
Potentially affect structure, function or is immunogenic
Typically done enzymatically but may be done chemically
proteases for tag removal
TEV Protease
Enterokinase
Thrombin
Self-cleaving tags
problems with removing a fusion tag
Protein of interest may also be cleaved
Harsh chemical conditions denature proteins
Tag removal less than 100% efficient
Poly-his tag triggers aggregation
May require additional separation steps
general protein stabilizers
Reducing Agents
Protease Inhibitors
Bulk proteins (e.g., BSA)
Amino acids (G, T, A, K)
Carbohydrates – reduce waters of hydration
Glycerol
Polymers (e.g., PEG)
specific protein stabilizers
Substrates
Ligands
Antibodies
lyophilization
drying of proteins
Common AA in alpha helix
AKREQ
AA not found in alpha helix
TIP
Kinases
Put on phosphate
De phosphorylates/phosphotases
Take off phosphate
What proteins are phosphorylated
Intercellular
Physical ways to degrade proteins
High temps
Freeze thaw
Agitation
Extreme pH
Biological ways to degrade proteins
Carbohydrases
Phosphotases
Proteases
Maintain protein stability
Keep in buffer
Minimize processing times
High purity reagents
Store frozen
Stable pH
