Biochem 285 Exam 1 Mearls Review

bonds that contribute to secondary structure

hydrogen bonds

atoms responsible for making hydrogen bonds

hydrogen atoms covalently bonded to highly electronegative atoms like oxygen (O), nitrogen (N), or fluorine (F)

bonds that contribute to tertiary structure

ionic bonds, hydrogen bonds (or other dipole-dipole bonds), dispersion forces, and disulfide bonds

difference between secondary and tertiary bonds

secondary structure bonds form exclusively between atoms in the peptide backbone, while tertiary bonds tend to form between the R-groups

how heat and treatment with SDS help to disrupt these bonds and denature a protein

Heat helps to disrupt the non-covalent interactions within a protein structure because bond formation is temperature dependent

SDS is amphipathic with a large non-polar portion and a polar charged portion. This allows it to interact with the different non-polar/polar parts of the protein and prevents it from refolding

domain

It may have a specific function within a protein.

It can fold independently from the rest of the polypeptide sequence of the protein.

True or False: Molecular chaperones require ATP to function

True

You have a protein that can be detected with a commercially available antibody. Which technique would allow you to detect and visualize the presence of this protein in your sample

Western Blot

rotein folding is considered favorable/spontaneous because the water surrounding the protein has more possible configurations and thus has higher _______

entropy

Protein folding is mostly driven by

the Hydrophobic Effect

What is a function of a protein chaperone

Stabilizes proteins that haven't folded yet or that have misfolded and allows to

them fold

during protein folding:

entropy of the water ______

entropy of the protein _______

Entropy of the water increases

Entropy of the protein decreases

A protein is considered "denatured"

The molecular interactions that give the protein its shape have been disrupted

treatments most often used to denature proteins

Heating to 100 ºC

Adding a high concentration of urea

Adding a detergent such as SDS

Which of the following techniques may provide you with a way to isolate your protein

Ion exchange chromatography

Size exclusion chromatography

2D gel

studying a protein that you think binds a specific DNA sequence

Purify the protein and split it into two samples. Mix one sample with DNA and leave the other one alone. Run both samples through a Native-PAGE. Compare the migration patterns between the two samples.

SDS is a useful denaturant because

It is amphipathic (has polar and non-polar parts)

It binds to the non-polar parts of proteins, which prevents them from re-associating via hydrophobic effect.

It can interact with ionized functional groups

It can disrupt hydrogen bonds

The spinning allows the egg white proteins the chance to refold correctly. What does the urea do?

It breaks up the aggregated egg white protein by destabilizing the H-bonds thatformed during cooking.

Explain what characteristics of the protein and its ligand provides specificity ofbinding (i.e. what dictates the Kd of a protein-protein or protein-ligand interaction?)

The architecture of the bonds, as well as the number of bonds, formed between the atoms on the ligand and the atoms on the amino acids in the binding pocket of the protein

Which types of chemical bonds typically hold the protein and ligand together?

Non-covalent bonds like hydrophobic interactions, van der Waals, hydrogen bonds and electrostatic (ionic) bonds.

α-1-Antitrypsin Deficiency disease is caused by a single codon change, resulting in a substitution of a lysine for a glutamic acid. Consider the chemistry of these two different amino acid residues. Why would such a change be particularly detrimental to the proper folding of the polypeptide given the knowledge that the arginine was forming an ionic bond (electrostatic interaction) with the glutamic acid?

The arginine is positively charged and the glutamic acid is negatively charged. Theglutamic acid was originally forming an electrostatic interaction. The change to lysinewould mean that two positively charged R groups are trying to sit in space near eachother. They would repel each other and disrupt the folding of the protein.

A nuclear import peptide sequence can occur anywhere (inside or outside) on a folded protein, and most often is made up of a string of charged amino acids such as -Lys-Lys-Lys-Arg-Lys-

FALSE; It is true that a nuclear import peptide typically has a string of basic amino acids, but they must be present on the SURFACE of a protein, not anywhere.

GEF adds a phosphate to GDP to make GTP

FALSE; GEF triggers the release of GDP from a G-protein to allow GTP to bind in its place

A protein requires a signal sequence to be retained in the nucleus

FALSE; it requires a signal to be imported and exported, but not retained

One of the ways that glycosylation is used in the ER is to mark proteins for sorting to other cellular compartments

TRUE; the sugar groups that are added during glycosylation are continually modified as the protein is made in the ER and passed through the endocytic/secretion pathways.

Explain the use of chaperones in protein translocation into the mitochondria

Chaperones bind to mitochondrial proteins as they are synthesized in the cytosol and prevent them from fully folding. When the protein is translocated into the mitochondria, chaperones in the matrix bind the incoming peptide and help it come through the translocator, and then help it fold properly

Explain the role of GTPases in nuclear transport

GTPases are proteins that bind and hydrolyze GTP to GDP. Binding GTP/GDP changes their conformational shape and activity. In the case of nuclear transport, a GTPase called Ran is loaded with GTP after interacting with GEF. This activates Ran and allows it to bind its ligand(importin/exportin). After transport, GAP then stimulates Ran to hydrolyze GTP to GDP, and this changes the conformation of Ran so that it releases its ligand.

Proteins with an N-terminal ER signal sequence are usually trafficked to the ER via SRP, butproteins with a C-terminal ER signal sequence may be trafficked to the ER by a differentmechanism. Explain why SRP is most strongly associated with N-terminal ER signal sequences

SRP binds to an N-terminal signal sequence just as this signal peptide emerges from the ribosome, simultaneously pausing translation of the protein until the ribosome can be brought to the ER sec61 translocon. Thus, an important feature of SRP is that it pauses translation before the protein is fully synthesized. If an ER signal sequence were located on the C-terminal end of a protein, it will likely be too late for SRP to pause translation before the protein is synthesized. In this case, it may be important for additional proteins, like molecular chaperones to bind the unfolded protein and help traffic it to the translocon. This would reflect a post-translational translocation mechanism as opposed to the SRP driven co-translationaltranslocation process

Explain how transmembrane proteins are made

A transmembrane protein will be initially targeted to the ER through a hydrophobic signalsequence. This signal sequence is inserted into the ER translocator channel. As it is made, hydrophobic patches in the peptide are recognized as transmembrane spanning regions, and the transfer is paused so that the Sec61 translocator can release that part of the peptide chain into the membrane. The protein continues to be synthesized and is oriented in the membrane based on the presence of charged amino acids that flank the hydrophobic regions.

Would the N-terminus still undergo N-linked glycosylation (represented by bluehexagons)?

It would not, because the N-terminus is now in the cytosol. N-linked glycosylation occurs in the ER

what would happen to nuclear transport in yeast strain that had a loss of function mutationin Ran-GEF? Nuclear Import

Ran-GEF protein converts Ran-GDP to Ran-GTP in the nucleus. Ran-GTP can then go on to interact and change the conformation of importin. This conformational change allows import into dissociate from its cargo which will remain in the nucleus. If Ran-GEF is mutated, cargo will not be released from importin

what would happen to nuclear transport in yeast strain that had a loss of function mutationin Ran-GEF? Nuclear Export

During nuclear export, Ran-GTP helps the exportin protein to bind to cargo and facilitates the exportin-cargo export from the nucleus to the cytoplasm. If there is a mutation in Ran-GEF, Ran-GTP will not be made and exportin will not be able to properly bind to its cargo for export.

Post-translational modifications refer to

The formation of covalent bonds between protein subunits

The addition of branched carbohydrates to a protein

A secreted protein, once translated, will follow which of the following pathways through the cell?

Cytosol, ribosome, ER, Golgi, vesicle, PM (plasma membrane)

ER, vesicle, Golgi, vesicle, PM

ER, ribosome, cytosol, vesicle, Golgi, PM

Cytosol, vesicle, Golgi, vesicle, PM

ER, cytosol, vesicle, Golgi, PM

ER, vesicle, Golgi, vesicle, Plasma Membrane

What would happen to nuclear transport if Ran were bound to a non-hydrolysable form of GTP (GMP-PNP)?

Exportin would be exported from the nucleus

Exportin would never be released in the cytosol

Ran-GAP would become ineffective

You have made a mutation in Ran. This mutation causes Ran to have a much higher affinity for GDP than GTP. What will occur as a result?

Higher concentrations of Ran-GDP in the nucleus

A lower rate of nuclear export

Cell death

What would be the consequence of eliminating all Ran-GEF from the nucleus of a cell?

Nuclear transport receptors would be unable to release their cargo in the nucleus

True or False: For Ran, enzymes for GTP hydrolysis to GDP are on the nuclear side of the nuclear envelope, and enzymes for the exchange of GDP for GTP are on the cytoplasmic side

False

True or False: Activity of nuclear transport receptors is regulated by Ran, a GTP binding protein

True

True or False: Proteins that must enter the nucleus have amino acid sequences called nuclear localization signals

True

True or False: Nuclear localization signals are recognized by nuclear transport receptors called importins

True

True or False: Nuclear localization sequences are recognized by nuclear transport receptors which direct transport of the proteins through the nuclear pore complex

True

In co-translational translocation, the primary force that drives the polypeptide through these c61 translocator comes from _______

the ribosome

A transmembrane protein has an N-terminal ER signal sequence and one Stop Transfer Anchor (STA) sequence. What is the orientation of the protein in the membrane once it is fully translated?

The N-terminus is in the ER, and the C-terminus is in the cytoplasm

Which of the following might you expect to find in (or on) most eukaryotic cytosolic proteins? (Select all)

disulfide bonds between cysteines

a hydrophobic interior and a hydrophilic surface

N-glycans attached to the surface of the protein

Alpha helix and beta strand/sheet secondary structures

a hydrophilic interior and a hydrophilic surface

A hydroPHOBIC interior and a hydroPHILIC surface

Alpha helix and beta strand/sheet secondary structures

Why are so many molecular chaperones found in the ER?

The ER is where many proteins are trying to fold in close proximity.

Some molecular chaperones help with protein translocation.

The primary structure ofa protein is determined by the _____________________

identity of the amino acids in the peptide sequence

A peptide bond is stronger than a single covalent bond because it has a partial double bond character. This means that not only is this bond stronger than a single bond, but it can also limit the geometry of the molecule because ___________

The rotation of the bond is limited due to resonance

Explain how you would categorize an amino acid based on its structure

Find the alpha amino and alphacarboxyl groups and identify the central carbon.

From the central carbon, identify the side chain.

Examine the side chain to see if it has an amine or carboxylic acid functional group.

If so, it belongs in the basic or acidic groups, respectively (unless there is also a large hydrocarbon component - see Tryptophan(W)).

If it is not an acidic or basic amino acid, determine if it is polar by looking for hydroxyl or amide functional groups. Note: Tyrosine (Y) has a -OH, but can also be considered non-polar because if its large aromatic ring.

If it is not acidic, basic, or polar it is likely non-polar. Non-polar amino acids have mostly hydrocarbons in their side chains.

A few tricky ones include Tryptophan (W), Cysteine (C), Proline (P), and Tyrosine (Y). These can all be categorized as non-polar, even though C, P, and Y sometimes behave in a more polar way in a protein.

α-1-Antitrypsin Deficiency disease is caused by a single codon change, resulting in a substitution of a lysine for a glutamic acid. Consider the chemistry of these two different amino acid residues. Why would such a change be particularly detrimental to the proper folding of the polypeptide given the knowledge that the arginine was forming an ionic bond (electrostatic interaction) with the glutamic acid?

The arginine is positively charged, and the glutamic acid is negatively charged. Theglutamic acid was originally forming an electrostatic interaction. The change to lysinewould mean that two positively charged R groups are trying to sit in space near eachother. They would repel each other and disrupt the folding of the protein.

Glycine and proline are known as "helix breakers". Explain why

Both amino acids disrupt the regular hydrogen bonding pattern essential for alphahelical structure.

Glycine's R group is very small and rotation around the phi and psibonds are quite flexible. This flexibility makes it harder for the atoms to "settle" into the position conducive for H-bond formation.

Proline's R-group is covalently bound to the imide nitrogen, which means that rotation in the phi-psi bond space is severely restricted.

Consequently, proline is unable to complete the H-bonding chain of the helix and it will cause a "kink" in the alpha helical structure.

The model below reflects the molecular chaperone hsp70. What information would you need to determine if the blue and brown coloring of the model represents domains or subunits?

It would help to know where the N and C terminus are (or termini, if more than one folded polypeptide is present) so you could determine how many folded polypeptides were in the structure. If it were one polypeptide, you could infer that this protein was a monomer, and the coloring was meant to show the domains within that monomer. If it were two polypeptides, you would likely conclude that the coloring represented subunits. Hsp70 is created from one polypeptide, so in this model we are looking at the two domains found within the monomer.

Describe a key difference between covalent and non-covalent bonds.

The atoms in a covalent bond share some of their electrons, but the atoms in a non-covalent bond do not share any electrons.

Which term describes ALL types of noncovalent bonds?

electrostatic

London dispersion forces are attractive forces that arise when ___________

oppositely charged dipoles, created through the random distribution of electrons around their atoms' nuclei, interact.

True or False: Dipole-dipole interactions must involve a hydrogen atom.

False

True or False: Hydrogen bond formation is possible between -CH and-NH3

False

True or False: The carboxylic acid (COOH) functional group could form hydrogen bonds with water.

True

True or False: An ionic bond could form between two carboxylate (COO- ) functional groups

False

True or False: Hydrocarbon chains can interact through dispersion forces

True