the ability of a protein to function is determined by its ability to ___
interact and bind to other molecules
specificity vs affinity
specificity- shape of binding site/ active site complements the shape of the ligand, allowing for non-covalent interactions
affinity- strength of the non-covalent interactions ; can tell by the overall number of interactions. more interactions = more affinity
what are the 2 models for specificity?
1) hand - in -glove model where the shape of the active site and the ligand match up
2) induced fit model: loosely bound ligand interacts with the substrate to conformationally change it so that it fits better in the active site
Describe affinity and specificity as it relates to antibody-antigen interactions. what is an antibody?
- antibodies consist of a tetramer with 2 heavy H chains and 2 light chains with constant and variable regions
- the variable region enables antibodies to recognize and bind.
- this region is responsible for specificity because it recognizes the correct aa sequence
- also 1 antibody = 1 antigen is a form of specificty
- affinity comes from the variable region binding to the aa ** the variable loops provide increased surface area which allows for more non covalent interactions which equals more affinity
Proteins interact with each other in 3 basic ways: list and describe with examples
1) surface-string:
surface has groove that another ligand can fit into
ex: SH2 of sarcoma can bind to phosphorylated tyrosine bc the surface of the SH2 motif forms a groove or cleft with the phosphorylated tyrosine
2) helix-helix
same as coiled-coil motif bc helices interact through hydrophobic interactions (remember the leucine zipper example) btwn the alpha helixes
3) Surface-surface
R groups match up and form non covalent interactions
proteins can be modified and this can have significant impacts on their…
function
how does phosphorylation affect protein function?
- it is a common post translational modification
- can change the charge and the type of binding the protein can do
ex: protein kinase will phosphyorylate a serene which is a polar aa (can form H bonds).
- no w the serene has increased size, and forms ionic bonds instead of H bonds
-phosphorylation can also turn proteins on/off
two examples of how phosphorylation affects protein function were given in class.
It will probably be tested, so describe them both
1) MAP kinase:
- in inactive kinase, it is in an open state and threonine and tyrosine (remember these along with serine are the 3 aa that can be phosphyorylated) are unphosphorylated
- kinase comes in and phosphorylates the 2 residues—this turns the protein on. Now the conformation changes and the kinase can form a dimer that binds ATP AND the appropriate ligand
- active site now keeps ATP in and activates it
2) SRC KINASE:
- tyrosine residue in the carboxyl terminal TAIL of the protein is phosporylated in the OFF state
- remember that SH2 binds to phosphorylated tyrosine’s
- therefore the SH2 domain binds to the Src tail
to activate the Src we need 3 things:
1) phosphate on the tyrosine has to be removed by a phosphatase
2) the SH3 domain binds to an activating ligand to cause change in structure
3) ACTIVATION LOOP of the kinase domain needs to be phophorylated
THEN Src is active
why is there a fatty acid group at the end of the Src protein?
can associate with membrane for transmission purposes
Describe all the types of protein phosphyorylation- non covalent
1) farnesylation and myristylation:
addition of fatty acid or other hydrocarbon to a protein, usually to anchor the protein to the membrane
2) acetylation- can alter the way the histones bind to DNA
3) glycosylation: introduction of mono/oligo/poly saccharide as covalent components of proteins
- alters protein folding and the carbs can confer functions to the proteins
- usually proteins destined for plasma membrane
4) hydroxylation: addition of OH group
ex: seen in collagen (need vitamin C to do it)
describe the types of protein phosphoyrlation- covalent
addition of ubiquitin:
- mono: usually regulation by adding 1 ex: historegulation
- multio: usually regulation as well by adding a chain, but not necessarily ex: endocytosis
-poly: can be for degrading proteins or DNA repair fans forming a scaffold
describe in more detail the degradation by ubiquitin
Ub chains are recognized by proteosome cap which has DUBs or deubiquitinases
the DUBs recycle the ubiquitin and control the length of the chain allowed.
- protein targeted for destruction can bind to the cap, then feed into the proteosome core of the machine.
- proteases inside cleave the protein and they are recycled or fed into the antigen pathway.
Describe the technique for protein characterization by size. is it diagnostic or discovery?
chromatography separates by size:
-column matrix has beads that smaller aa go through and spend a lot of time getting through the nooks and cranes
- the longer aa will go through the sides, going faster
- diagnostic to purify proteins
describe ion exchange chromatography. what can it be used for?
what about an affinity column?
separate proteins based on charge:
-if you put in positive beads, then the negative ones will flow through quickly but the positive ones will stick
affinity columN:
- molecules are covalently bound to the beads to find out interaction with protein of interest
ex: glucose - take off sticky ones by adding glucose (if glucose column) or salt for ionic columns
describe SDS page. what does it stand for?
Sodium dodecyl sulfate polyacrylmade gel electrophoresis
- SDS (detergent) is added to unfold/denature the protein and coat with a negative charge
- this eliminates the R group/ neutralizes them all to a negative charge
- the small things travel faster and larger things travel slower
-separation based on weight and antibody binding specificity
after SDS page, what can be done to the purified protein?
- generate antibodies
- mass spectrometry to identify post translational modification
- analsysis of structure and biochem properties
describe a western blot analysis. when is it best to use this? is it diagnostic or research? what is a clinical example
SDS page used for western blot
- take gel and transfer to it a membrane
- incubate with antibody
- add label so you can detect it
- react with substrate
-therefore asses levels of specific protein by using label after gel electrophoresis
*best for 1-2 proteins
both diagnostic and research
example: used to confirm HIV serostatus
describe a 2 dimensional gel electrophoresis. when is it best to use this? is it diagnostic or research? what is a clinical example
best for hundreds of proteins
usually discovery and not diagnostic bc hard to duplicate
1) first dimension: separate proteins by charge (not SDS but another detergent) on an isoelectric focusing (IEF ) gel that has a pH gradient so that protein will migrate to where charge is zero
2) Second dimension: run on a SDS PaGE gel to see if a protein is phosphorylated as well. Each protein moves differently through the gel matrix depending on its size. The separation of proteins is distributed roughly according to size (molecular weight), like other electrophoresis methods.
describe mass spectrometry
used for thousands of proteins at a time
1) degrade with protease
2) run on mass spec
3) get separation by size and very accurate measure depending on time of flight
-correlate to human genome and also know if phosphorylated by its flight time
Thepatientdescribedinquestion8,whohas multiple myeloma, has not responded to numerous treatments, and his disease is pro- gressing. He sees his oncologist, who wants to start him on the drug bortezomib. Bortezomib inhibits the proteasome from degrading pro- teins. Which class of intracellular proteins will not be specifically degraded as a result of taking this drug?
TheanswerisC. : Polyubiquitinated proteinsTheproteasomenormallydegradesproteinsthathavebeenpolyubiquitinated. As such, in the presence of bortezomib, polyubiquitinlated proteins will accumulate within cells, leading to a selective adverse effect on the cancer cells (myeloma cells) because these are the cells growing most rapidly. Proteins with PEST sequences are rapidly degraded by nonspecific intra- cellular proteases. Although the immunoglobulin light chains are forming the amyloid proteins in this disease, these structures are difficult to degrade, such that inhibiting the proteasome has no effect on the degradation of the amyloid proteins. Immunoglobulin heavy chains are not accu- mulating in this disorder.
domains are a type of ___ structure
Domains are elements of tertiary struc- ture.
3.3 Which one of the following statements concerning the binding of oxygen by hemoglobin is correct?
A. The Bohr effect results in a lower affinity for oxygen at higher pH values.
B. Carbon dioxide increases the oxygen affinity of hemoglobin by binding to the C-terminal groups of the polypeptide chains.
C. The oxygen affinity of hemoglobin increases as the percentage saturation increases.
D. The hemoglobin tetramer binds four molecules of 2,3- BPG.
E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons (H+).
Correct answer = C. The binding of oxygen at one heme group increases the oxygen affinity of the remaining heme groups in the same molecule. Carbon dioxide decreases oxygen affinity because it lowers the pH; moreover, binding of carbon dioxide to the N-termini stabi- lizes the taut, deoxy form. Hemoglobin binds one molecule of 2,3-BPG. Deoxyhemoglobin has a greater affinity for protons and, therefore, is a weaker acid.
- higher pH means more basic!!!
