Exam 2

A protein that is a tetramer with four identical subunits of 10 kD each would show major bands on a properly run SDS-PAGE gel at

10 kD

A mixture of five proteins are analyzed by MALDI-TOF. Their molecular weigths are 3 kD, 22 kD, 100 kD, 225 kD and 515 kD. A peak appears in the mass spectrum at 33.3 kD. Which protein most likely gave this peak?

100 kD

In the 2D gel image below, the red spot represents and unmodified version of a protein with a molecular weight of 150 kD. Which grey spot is appropriately placed to represent a version of that protein that has been phosphorylated at a serine reside (a phosphate group has been covalently attached to the serine hydroxyl group)? The SDS-PAGE portion of the gel spans from 10 kD to 2000 kD. 1 2 3 4

4

You are purifying a protein that you think will win you the Nobel Prize someday. If you started with 15,000 mg of total protein in your homogenate with 150,000 units of total activity, then after two steps of purification you have 9750 mg of total protein and 68602 units of total activity, what is the percent yield after those two steps? Report your answer as a percentage to the nearest whole percent (56%, for example).

46

A tetrapeptide contains alanine, lysine, glutamate, and methionine. If cyanogen bromide cleaves the tetrapeptide into a tripeptide and free glutamate, and trypsin cleaves the tetrapeptide into two dipeptides, what is the sequence of the tetrapeptide? AKME AKEM MAKE KAME EMAK

AKME

A sample of blood proteins is separated on a 2D-gel. If the patient is young enough to express significant amounts of both fetal and adult hemoglobin, and fetal hemoglobin has less positive charges than adult hemoglobin but the sizes are nearly identical, where would they be found on the gel relative to one another?

At the same position in the SDS-PAGE dimension, but fetal hemoglobin will be found at lower pH in the isolectric focusing dimension

In ion exchange chromatography using diethylaminoethyl (DEAE) cellulose, which of the following amino acids would elute last?

D

If a protein has an isoelectric point (pI) of 3.2, which of the following amino acids is it likely to have a lot of?

D and E

The N-terminal sequence of a peptide can be determined by

Edman degradation

NMR determination of protein structure requires that proteins be crystallized into ordered structures.

FALSE

"Hemoglobin is a tetramer, but myoglobin is a dimer."

False

All mass spectrometers include an arc magnet to select certain ions.

False

GFP is an antibody.

False

IMAC chromatography separates molecules based on size.

False

In affinity chromatography, molecules are separated based primarily on their charge.

False

The T-state of hemoglobin binds oxygen more tightly than the R-state.

False

When a protein is 'salted-in', it precipitates and is removed form a solution.

False

Book: Protein sequencing1. Determine the sequence of hexapeptide on the basis of the following data. Note: When the sequence is not known, a comma separates the amino acids Amino acid composition: (2R, A, S, V, Y) N-terminal analysis of the hexapeptide: A Trypsin digestion: (R, A, V) and (R, S, Y) Carboxypeptidase digestion: No digestion. Chymotrypsin digestion: (A, R, V, Y) and (R, S)

First amino acid: ALast amino acid: R (not cleaved by carboxypeptidase)Sequence of N-terminal tryptic peptide: AVR (tryptic peptide ends in K)Sequence of N-terminal chymotryptic peptide: AVRY (chymotryptic peptide ends in Y)Sequence: AVRYSR

If a protein has an isoelectric point (pI) of 10.2, which of the following amino acids is it likely to have a lot of? M and W D and E C and P H and R W and Y

H and R

Which of the following proteins will elute first (go through the fastest) in size exclusion (also called molecular exclusion) chromatography?

Protein X (Molecular weight 149,000 g/mol, charge +1)

In the absence of oxygen, hemoglobin heavily favors the

T-state

Cyanogen bromide will cleave the peptide KWNEMDAR after the K after the M after both the E and the D after the W actually, it won't cut it at all

after the M

To make a monoclonal antibody, as opposed to a polyclonal antibody,

an antibody producing spleen cell is fused to a cancer cell

In 'Sandwich ELISA' the meat is _____________ and the bread is ______________.

antigen; antibody

Antibodies bind to

antigens

Which of the following amino acids has its isoelectric point at the lowest pH? aspartic acid glycine valine arginine methionine

aspartic acid

Antibodies are useful because they

bind things

A quick way to start purifying a protein is to first fractionate cellular organelles and collect the one with the protein of interest. If you want to collect fractions that separately contain nuclei, mitochondra, endoplasmic reticulum, and cytosol, how is this most easily done?

centrifugation

In ion-exchange chromatography, molecules are separated based on

charge

Chymotrypsin degrades proteins from the N-terminus one amino acid at a time cleaves proteins after positively charged amino acid residues is another name for cyanogen bromide cleaves proteins after large hydrophobic amino acid residues cleaves proteins after methionine residues

cleaves proteins after large hydrophobic amino acid residues

Dicyclohexylcarbodiimide is used to

couple amino acids (covalently bind them to each other)

In determining the following structure by NMR, the structure below would be specified by

crosspeaks in 2D NOESY spectra between protons 2 and 5

A reagent that selectively cleaves after methionine residues is

cyanogen bromide

ELISA is a method that is used to

detect and quantify proteins and/or small molecules

A method of exchanging the buffer (and other small molecules) present in a solution of protein is to use a semipermeable membrane that lets only the small molecules through. This technique is called

dialysis

A common reagent that activates amino acid carboxylates for solid phase protein synthesis is

dicyclohexylcarbodiimide

In protein x-ray crystallography, the crystal essentially serves as a

diffraction grating

In protein structure determination by NMR, the NMR provides

distance constraints

When determining the structure of a protein by NMR the NMR experiments themselves primarily provide

distance constraints

Fetal hemoglobin has a lower affinity for oxygen than adult hemoglobin.

false

In SDS-PAGE, the SDS serves to give proteins constant charge/mass denature proteins, so shape doesn't affect the separation preserve protein structure by stabilizing hydrphobic interactions give all proteins the same mass give proteins constant charge/mass and denature proteins

give proteins constant charge/mass and denature proteins

Which of the following amino acids has an isoelectric pH of 3.2? lysine glutamic acid asparagine methionine glycine

glutamic acid

An antibody

has two antigen binding sites

This graph of hemoglobin oxygen saturation clearly shows that

hemoglobin has a lower affinity for oxygen when the pH is low or the carbon dioxide concentration is high

Myoglobin's primary role in the body is to

hold a reserve of oxygen for muscle that is employed when oxygen levels become dangerously low

A student is carrying out a sandwich ELISA. Their first step should be to _______________.

immobilize monoclonal antibody in the wells of a 96-well plate

2,3-BPG modulates the oxygen affinity of the hemoglobin tetramer (shown) by binding

in the center of the tetramer, where it can contact all of the subunits

The Edman Degradation

is a method for sequencing proteins from the N-terminus

The 2,3-bisphosphoglycerate (2,3-BPG) binding site in hemoglobin

is in the center of the tetramer, contacting all four domains

Unlike the oxygen binding curve (oxygen saturation versus partial pressure of oxygen) for hemoglobin, the curve for myoglobin

is not sigmoid at all

A major benefit of the sigmoid oxygen binding curve (oxygen saturation versus partial pressure of oxgyen) for hemoglboin is that

it increases the amount of oxygen transferred from lung to oxygen deprived tissue

Fetal hemoglobin needs a much higher oxygen affinity than adult hemoglobin. This is accomplished by expressing a slightly different gene in the fetus. This fetal gene has a higher oxygen affinity because

it will not bind 2,3-BPG

When centrifuging sub-cellular structures, what organelles pellet out at 10,000 x g?

mitochondria

One difference between hemoglobin and myoglobin is

myoglobin is monomeric, whereas hemoglobin exists as a tetramer

A reagent that oxidizes cystines (cysteine disulfides) into cysteic acids that can no longer form disulfide bonds is beta mercaptoethanol performic acid vitamin C cyanogen bromide dithiothreitol (DTT)

performic acid

Which is easier to produce?

polyclonal antibodies

Which of the following proteins would elute first from a size exclusion chromatography column?

protein Z (MW 250,000 g/mol; charge +3)

The main drawback of protein X-ray crystallography is that

proteins must first be crystallized and conditions may never be found to crystallized the protein of interest

Dialysis is carried out on a solution containing a 100 kD red protein, a 10 kD yellow protein, and a 2,000 kD blue dye by placing the mixture in the dialysis bag. If the dialysis bag has a pore size of 50 kD, what color will the solution in the bag be after several equilibrations and buffer exchanges?

red + blue = purple

Dithiothreitol (DTT) is used to

reduce disulfide bonds

Dialysis

separates small molecules from large molecules

Gel-filtration chromatography largely separates proteins (or other molecules) based upon

size

In normal SDS-PAGE, separation is based on

size

Cyanogen bromide's specificity for cleaving amide bonds after methionine residues results from the formation of a required ______________ intermediate

sulfonium salt

In the Edman Degradation, phenyl isothiocyanate reacts with ___________________.

the N-terminal amino acid of a peptide

In the Edman Degradation, phenyl isothiocyanate reacts with ___________________. methionine residues in a peptide Edman, dissolving him completely (poor fellow!) the C-terminal amion acid of a peptide cysteine sidechains the N-terminal amino acid of a peptide

the N-terminal amino acid of a peptide

Which of the following is not an applicaiton of antibodies?

the Nuclear Overhauser Effect

In the MWC model of cooperativity as applied to hemoglobin, at very low oxygen pressure

the T state is strongly favored

To purify an enzyme, liver cells are homogenized in a Dounce tube and centrifuged at 500g for 10 minutes. The supernatant is centrifuged further for 10,000g for 20 minutes, then the resulting supernatant is centrifuged at 100,000g for one hour. The resulting pellet is resuspended in buffer and used for enzyme kinetics. The enzyme being studied must be found in

the endoplasmic reticulum

To purify an enzyme, liver cells are homogenized in a Dounce tube and centrifuged at 500g for 10 minutes. The supernatant is centrifuged further for 10,000g for 20 minutes, then the resulting supernatant is centrifuged at 100,000g for one hour. The resulting pellet is resuspended in buffer and used for enzyme kinetics. The enzyme being studied must be found in the outer mitochondrial membrane the nucleus the endoplasmic reticulum the cytosol the inner mitochondrial membrane

the endoplasmic reticulum

If oxgyen leaves hemoglobin and takes an electron with it, oxidizing the iron(II) to iron(III),

the oxygen becomes superoxide

2,3-Bisphosphoglycerate binds to

the positively charged hole at the center of the hemoglobin tetramer

In MALDI-TOF mass spec, the mass selector is

the time it takes to traverse a flight tube

One ofthe benefits of allosterism in hemoglobin, which results in a sigmoid oxygen binding curve,is that

there is maximal change in oxygen binding between the lung oxygen pressure and the typical(or active) tissue oxygen pressure

The heme in hemoglobin is held in the enzyme (in part) by a ligand interaction between the heme iron and

a histidine in the protein

Advances in CryoEM depend significantly on advancements in computing.

True

Advances in computers have increased our ability to image proteins in cells at very high resolution.

True

Advances in techniques for the determination of protein structures by Cryo Electron Microscopy won the Nobel Prize in 2017.

True

Denatures protein will usually not bind to an affinity column as well as normal, folded protein.

True

Mass spec can be used to detect cancer cells.

True

Peptides can be synthesized using the techniques of Organic Chemistry.

True

Some nuclear hormone receptors wait in the cytosol for their hormone to arrive.

True

When we purify protein, we are going against the natural tendency toward disorder (we are fighting against entropy).

True

You can sequence a protein using mass spec (along with some other tools, like enzymes to cut the protein into smaller pieces).

True

When the protein bands in an SDS-PAGE gel are transferred to paper and stained with antibody, it is called a

Western Blot

Explain in a few sentences how a protein structure is determined by Nuclear Magnetic Resonance Spectroscopy (NMR). What is the role of the off-diagonal cross-peaks in 2D-spectra?

this technique depends on the fact that certain atomic nuclei are intrinsically magnetic. the property is called spin and the spinning of a proton generates a magnetic moment and this can be in either one of two orientations called spin states. the spin states can be used to determine the chemical surroundings of the hydrogen nucleus. Due to the electrons flowing around a magnetic field, this causes a magnetic field that opposes the applied field. If the magnetic field is kept constant and changing the frequency of the electromagnetic radiation (known as chemical shift), a resonance spectrum can be formed. Then off diagonal peaks or cross peaks show the information that that identifies the proton pairs that are 5 A apart. The 2D spectrum known as the NOSEY shows short proton distances and thus a 3D shape of a protein can be made using these distance relationships. The structures are calculated that the protons must be separated by by less than 5 A based off the NOSEY spectra and then by this, if enough distance is applied the 3D structure can be determined

The role of dicyclohexylcarbodiimide in peptide synthesis is

to activate carboxyl groups, giving them a better leaving group

The most common method used to determine the sequences of proteins today is

translation of DNA sequences in computers using the genetic code

a TOF mass spec

uses the time of transit through a flight tube as the mass selector

Immobilized metal affinity chromatography (IMAC) will retain proteins

with 4-6 consecutive histidine residues

Hemoglobin is a(n)

α2β2 tetramer