Exam 2017-03-12 Flashcards
Hydropathy plot, draw one and explain what it is
A plot that shows the hydrophicity of a protein, positive values mean high hydrophicity and negative values hydrophilic and is given for each amino acid so it will look like a graph that goes up and down over a line of 0 that represents each amino acid
b) Greek key motif, describe and draw
It’s 4 antiparalell betasheets connected by short loops,
whenusing Affinity chromatography with his tag what is added after puirification to release from stationary phase?
Is a chromotgaphy method used to purify proteins. It has a stationary phase of a ligand that binds to the protein of choice and a mobile phase. The target protein will bind to the stationary phase and the rest will be removed with the mobile phase. His-tag is a very common method that is a form of affnity chromotography where the proteins are genetically modified to have a his-tag that has high affinity for metal ions and is after the chromotagphy is done released from the stationary phase by adding imidazole
Western blotting
Western blotting is a technique that is used to detect a specific protein and measure the quanity detected. First SDS page is performed to seperate the proteins by size and then the proteins are transferred to a membrane. The membrane is blocked commonly by milk to prevent non speciifc binding to antibodies and then primary antibodies are added to bind to the protein of intrest. Then washing step is performed to unbound antibodies and then secondary anitbodies that are ften flourescnetly labelled is added that will bind to the primary antibody. Then detection is performed to see if any and how much the protein of intrest is found.
Integral membrane protein and peripheral membrane protein what they are and how they are to work with and an example of integral membrane proteins
Integral membrane proteins goes all the way through proteins and recive signals on the outside of the cell and cause a signal inside of the cell to be released. For example G-coupled receptor. They are hard to work with since they are hydrophobic and therefore can’t we soluble, they need the membrane for their structure so they are hard to pruify etc. Peripheral membrane proteins are only on the outside of the membrane attatched to the membrane. They are eaier to work with since they are more soluble and can be purifed using standard methods.
The transmembrane domain of GlpG folds by a two-state mechanism when the
protein is denatured using a denaturant. Draw a schematic graph of a two-state
folding/unfolding protein where [denaturant] is on the x-axe and the measured
parameter y (from a biophysical measurement) is on the y-axe.
It goes from folded to unfolded without an intermediate so there is folded transition state then unfolded it will look like an S curve.
Give the name of three different proteins that are important for in vivo folding
and describe their functions.
peptidyl prolyl isomerases (PPI) catalyze the cis-trans isomerization trans and cis configuration in proline residues. This accelerate the protein folding and correct misfolded proteins by binding to them and promotin refolding,
Protein disulfide isomerases (PDI), catalyze the formation of a disulfide bond between cysteine residues and can interact with misfodled proteins to either refold the protein or target it for degredation.
Chaperones or heat shock proteins such as GroES/GroEL are upregulated after heat shock in bacteria to assist protein folding but is also important for correct folding under normal cell conditions
Describe the technique of using equilibrium dialysis to monitor ligand
binding. Describe two other methods that can be used for a ligand binding assay.
The method of equilbrium dialysis is when you use a emipermable membrane to seperate two sides. Then you add the sample to one side and a ligand to the other side. The protein will follow the concentration gradient and diffuse to the other side of the semipermable membrane towards the lower concentration. Depending on the affinity of the ligand either it will bind to a lot of the ligand and there will be little free protein that will after a while reach equilbrium or a lot of free protein. Then the Kd can be calculated based on how much is bindign to the ligand. It is often measured using for example flourescense to measure how much is binding.
ITC is also another method to measure protein binding which is a method that measures heat released or absorbed during the binding process of the protein and ligand.
NMR takes quite long but can also be used and it uses a magnetic field to study the protein and the interaction with the ligand
Silk is an important textile material. -
Describe the secondary structure and tertiary packing structure of a model
of silkworm silk fibroin with the repetitive polypeptide sequence ….-Gly-Ala
Gly-Ala-Gly-Ala-
each peptide make a beta strand that is stacked together to make beta sheets ad due to glycine, alanine and serine all found a lot in silk being the smallest maino acids the sheets are packed very tightly. They have strong wanderwaals interactions and a hydrophobic core.
Different fibrous proteins have different means of covalent cross-link chemistry
between fibers to stabilize their structure.
Alpha-keratin is the major component of human hair (see Figure) and nails. How
is alpha-keratin cross-linked? (1p)
How is collagen cross-linked?
- In alpha keratine one in 9 amino acids are cysteine which make disphulfide bonds
- Keratins are composed of alpha helices in a coiled-coil motif.
- Collagen is high in proline and glycine and proline is a beta and alpha helix breaker so it has a triple helix structure. The prolines are what gives the cracks and turns and glycine is in the center.
- Thecross linking of collagen is the fomration of allolysine, through aldehyde condensationin cross linking. This is when lysine is oxidized by an enzyme to form allolysine which is then converted into an aldehyde. The aldehydes formed on the molecule then react with each other through condensation and this leads to the cross link of the collagen molecules
Biosynthesis of Collagen for e.g. bone is a highly regulated process. Describe the
pathway of biosynthesis of collagen in the cell starting with translation and
ending with collagen fibres
It begins with translation of mRNA in the ribosome in the cytoplasm. Collagen is synthesized as a long precursor molecule called procollagen. This precursor molecule contains three individual polypeptide chains, each encoded by a separate gene.
Procollagen undergo posttranslational modifications, such as hydroxylation of proline and lysine residues.
Then some hydroxolysine are glycolysated.
Then then form triple helix secondary structure with the help of chapereone proteins.
Last step is cross-linking between collagen molecules mediated by enzymes to form covalent bonds between lysine residues.
Protein aggregation is causing several chronic human diseases, such as
Alzheimer’s disease. In such diseases called amyloid diseases around 30
different proteins (each specific for each disease) have been identified form
insoluble amyloid fibrils in humans. -
What is the common secondary structure of proteins in amyloid fibril cores?
Beta-sheet conformation. In amyloid diseases like Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, proteins aggregate and form insoluble fibrils characterized by long, unbranched β-sheet-rich structures. These β-sheets stack together to form the core structure of amyloid fibrils, which are resistant to degradation and accumulate in tissues.
Many inherited diseases (e.g. Cystic fibrosis) are caused by protein folding
defects and are so called loss of function diseases due to degradation of
destabilized mutant protein. -
What are the two major pathways for protein degradation in the cell?
Ubiquitin-Proteasome System (UPS):
In the UPS pathway, proteins marked for degradation are tagged with a small protein called ubiquitin. Once tagged with ubiquitin, the protein is recognized and degraded by the proteasome. The proteasome disassembles the tagged protein into small peptides, which are further degraded into amino acids by cellular peptidases.
Lysosomal Degradation Pathway (Autophagy-Lysosome Pathway):
In the lysosomal degradation pathway, proteins and cellular components targeted for degradation are engulfed by double-membrane vesicles called autophagosomes. Autophagosomes fuse with lysosomes, which contain various hydrolytic enzymes. They break down the macromolecules into smaller components that can be recycled.
For a graph (chevron plot) with an unfolded protein that is let to refold and then adding increasing amount of denaturant unfolding again what does the slope of the curve tell us?
Steeper slope higher cooperativity and it folds/unfolds at a faster rate and less steep less cooperativity and unfolds/folds at a slower rate
In a chevron plot that starts with an unfolded protein that is let to refold and then adding increasing amount of denaturant unfolding again how did they achieve the unfolding and folding?
Start with a denatured protein could be denatured by heat for example in a buffer start with an unfolded protein with 0 concentration of denaturant and slowly add an increasing concentration o denaturant first it will start to refold and then as the denturant concentration increases it will denature again
