Biochemistry Exam 1

How to calculate percent yield on a protein purification experiment?

(current total activity/ original total activity)* 100

How to get amino acids given 5' to 3' DNA strand?

-5' to 3' strand 3' to 5' strand -transcribed mRNA -amino acid

Identify the true statements regarding disulfide bridges (disulfide bonds).

-A disulfide bridge forms between two cysteine residues. -Disulfide bridges can exist between two amino acid residues on the same chain. -Disulfide bridges have a stabilizing effect on proteins.

What happens in replication?

-DNA polymerase -deoxynucleoside triphosphate -primer

How many hydrogen bonds are formed between each base?

-Guanine and Cytosine form three hydrogens -adenine and thymine form two hydrogen

What classifies reactions as spontaneous (favorable)? unfavorable?

-If we have a Δ G that is less than zero, if it's negative, then that means it's spontaneous (favorable) -If we have a Δ G that is greater than zero, so positive Δ G, then that reaction is going to be nonspontaneous, not favorable in that direction.

What two main techniques are used to determine 3D protein structure? One word answers

-NMR -Crystallography

Select the true statements about SDS‑PAGE, a method of separating proteins. Assume that SDS‑PAGE is performed under reducing conditions.

-Proteins are separated in a polyacrylamide gel matrix. -Smaller proteins migrate faster through the polyacrylamide gel. -Protein‑SDS complexes have similar mass to charge ratios; therefore, separation is by size.

What is transcription?

-RNA polymerase -nucleoside triphosphate -promoter

Identify the statements that describe the structure of DNA.

-The five‑carbon sugar of DNA is called deoxyribose -Adenine is paired with thymine, and guanine is paired with cytosine. -A DNA double helix contains two sugar‑phosphate backbones oriented in opposite directions.

What is true of peptide bonds?

-They are generally in the trans and rarely in the cis configuration. -They contain an unusually long C−CC−C bond. -They tend to be planar. -Peptide bonds are amide linkages in which the nitrogen bears no charge

Which of the following statements accurately represents characteristics of NMR and x‑ray crystallography?

-X‑ray crystallography generally gives a more detailed (higher resolution) view than does NMR.

What is tRNA?

-anticodon -smallest RNA molecule -transports amino acids

What general statement describes affinity chromatography?

-can separate molecules based on protein-ligand binding -stationary phase has a covalently bound group to which a protein in the mobile phase can bind

What is mRNA?

-carries genetic information from nucleus to cytoplasm -codon

Describe nucleotides.

-contain base, monosaccharide, and phosphate group -monomers of nucleic acids -can be named deoxyguanosine 5'-monophosphate -found in RNA and DNA -contain base and monosaccharide -contain purine and pyrimidine

Describe structural features of purines.

-contain four ring nitrogen atoms -contain two heterocyclic rings -adenine and guanine

Describe structural features of pyrimidines.

-contain one heterocyclic ring -contain only two ring nitrogen atoms -cytosine, thymine, and uracil

What three steps are repeated during each cycle of solid phase DNA synthesis?

-coupling -oxidation -deprotect

What two steps are repeatedly performed in solid phase peptide synthesis?

-coupling (DCC) -deprotection (TFA)

What happen when a protein is treated with cyanogen bromide? trupsin?

-cyano= cuts the C-terminus of methionine -trypsin= hydrolyzes proteins at the carboxyl side of the amino acids lysine or arginine

A protein has been eluted from a diethylaminoethylcellulose (DEAE‑cellulose) column by washing the column with a buffered salt solution. Since salt can interfere with the next step of your protocol, which is protein quantification, this salt must be removed from your eluted sample. Which of the following will efficiently remove salt from your sample?

-dialysis -passage through a gel filtration column -dilution and concentration using a protein concentrator device

Describe nucleosides.

-dont contain phosphate group -are the product when a base bonds at C1 of ribose or deoxyribose -found in RNA and DNA -contain base and monosaccharide -contain purine and pyrimidine

Which two amino acids dont have standard Ramachandran plot and why?

-glycine (G) b/c chrial and H side chai -proline (P) b/c has 5-membered ring as aide chain so resticted

On a two-dimension gel electrophoresis, which proteins have the highest pI value? highest molecular weight?

-highest pI = highest pH -highest molecular weight will be located the highest (top) on the electrophoresis

In Proteins which can easily diffuse through a cells lipid bilayer, hydrophobic amino acids prefer to be on the _____ of the protein?

-inside (away from water surrounding). -outside of membrane is positive and inside is negative

Which of the two steps of two-dimensional electrophoresis has a final net charge of zero? negative?

-isoelectric focusing = 0 -SDS-PAGE= negative

_______________ proteins move faster on a gel filtration column. Explain

-larger -Because smaller proteins can penetrate into the beads more easily than larger proteins, they travel through a gel filtration column more slowly than larger proteins -In contrast, proteins migrate through the pores in an electrophoretic gel; thus smaller proteins move faster than larger ones.

Could a positive ΔH and negative ΔS ever result in a favorable reaction?

-not spontaneous forward -spontaneous reactions in reverse at all temperatures

Classify each protein example according to its highest level of protein structure. (primary to quaternary structure)

-primary= amino acid of myoglobin -secondary= C helix of a-lactalbumin -tertiary= single hemoglobin subunit with heme -quaternary= hemoglobin

What is Western blot with SDS-PAGE?

-proteins are denatured -proteins transferred to a membrane or sheet -can be used to detect a specific protein

What is ELISA?

-proteins are in their native state -can be used to detect antigen or antibody in a sample, depending on procedure -can be used to detect a specific protein

What statements are true about isoelectric focusing?

-proteins are separated by charge -carried out in a pH gradient -final net protein charge is zero

What general statement describes SDS- PAGE (discuss proteins)?

-proteins are separated in their denatured state -smaller proteins travel faster during separation -proteins move toward the positive electrode -proteins separated by size/ mass -final net protein charge is negative -carried out in a detergent solution

What general statement describes size-exclusion chromatography ( discuss proteins)?

-proteins are separated in their native state -larger proteins travel faster during separation -proteins of similar molecular weight but different can be separated -proteins separated by size

What is translation?

-ribosome -tRNA, mRNA, rRNA

What general statement describes ion-exchange chromatography?

-separate molecules by charge -stationary phase may contain negatively or positively charged groups

What general statement describes size-exclusion chromatography ( discuss molecules)?

-separated molecules by size -stationary phase contains cross-linked polymers with different pore sizes

Identify the applications for gel filtration chromatography.

-separation of components in a mixture by size -estimation of molecular weights

In what situation would a positive ΔH and positive ΔS result in a favorable reaction?

-spontaneous reactions above a certain temperature

On a two-dimensional gel of a mixture (SDS-PAGE), order the proteins in increasing elution volume on a size exclusion column.?

-the top of the SDS page will have smallest elution volume while bottom has largest.

What are the roles of sodium dodecyl sulfate (SDS) in two‑dimensional electrophoresis?

-to cause bound proteins to have a large negative charge -to denature proteins

Describe DNA polymerase.

-use deoxyribonucleoside triphosphates -semiconservative replication -needs primer

What general statement describes size-exclusion, affinity, and ion-exchange chromatography all have alike?

-use mobile phase and stationary phase to separate proteins

Describe RNA polymerase.

-use ribonucleoside triphosphates -conservative replication -does not need a primer

If the pI of a protein is 11 and the pH of the elution buffer is 7, will the protein be retained on a cation exchange column at low salt concentration?

-yes -When adding salt, proteins spend more time in the solution, the rate of their movement down the column increases dramatically, and proteins begin to elute from the column, usually in order of increasing charge

Monoclonal antibodies and polyclonal antibodies differ in the number of sites that they will bind on an antigen. How many sites on an antigen will a given monoclonal antibody bind to?

1

What is the maximum number of hydrogen bonds that one molecule of methanol as the hydrogen bond donor can form with water?

1

How many bases are there per turn in B-DNA?

10.5

How many amino acids are there per turn of an alpha helix?

3.4

What is the maximum number of hydrogen bonds that one molecule of acetic acid (CH3COOH) as the hydrogen bond acceptor can form with water?

4

DNA polymerase synthesizes DNA in what direction?

5' to 3'

Protein can be unfolded with a denaturant. At the concentration of denaturant that causes the protein to be "half unfolded" what percentage of fully folded protein will you normally have?

50

Which of the following is true regarding the processes of replication and transcription?

Although replication and transcription both require a DNA template, only replication requires a primer.

As a sample of DNA is heated, it is said to melt. What is occurring?

As hydrogen bonds are broken, the two strands separate.

What amino acids makes it difficult to determine the amino acid composition from the chromatograph?

Asparagine(N) and Glutamine(Q)

What six letters of the alphabet are not used as one letter codes for the twenty naturally occuring amino acids?

B, J, O, U, X, Z

Which restriction enzymes leave single stranded DNA after cutting (sticky end)?

BamHI, EcoRI, Hhai, XhoI

In Anfinsen's classic protein folding experiments what reagent did he use to reduce the protein?

Beta-mercaptoethanol

What do α helices and β sheets have in common?

Both are stabilized by hydrogen bonding involving carbonyl oxygens and amide nitrogens in the peptide backbone.

Which enzyme pairs corresponding nucleotides to a preexisting DNA chain in order to synthesize a new strand of DNA?

DNA polymerase

An important key to understanding the structure and function of DNA was the observation that the amount of A equaled the amount of T and the amount of G equaled the amount of C.This important observation was made by

Erwin Chargaff.

What is the bp recognition for EcoRI?

GAATTC

Which restriction enzymes do not leave any single stranded DNA after cutting (blunt end)?

Hae III

Of the 20 amino acids from which proteins are made, which is most likely to be present with its R group in a mixture of ionization states near physiological pH?

His

Which intermolecular force is directional?

Hydrogen bonds are among the strongest and most directional intermolecular interactions. -Van de Waals also has this?

How do monoclonal antibodies differ from polyclonal antibodies?

Monoclonal antibodies recognize only a single portion of an antigen molecule, whereas polyclonal antibodies recognize different parts of the antigen molecule.

In a solid phase peptide synthesis, which amino acid is added last, the N or C-terminal amino acid?

N-terminal amino acid

In solid phase peptide synthesis, which amino acid is added last, the N-terminal amino acid or the C-terminal amino acid?

N-terminal amino acid

What two techniques are used to determine the structures of proteins?

NMR Crystallography

If I cut a plasmid with one restriction enzyme and then my proposed insert with another enzyme will they ligate (join) together?

NO, they must be cut with the same restriction enzyme

Which of the following is true regarding the chirality of amino acids found in proteins?

Only L amino acids are found in proteins.

Which amino acid fits the worst in both an alpha helix and a beta sheet but best in a turn?

Proline (P)

What is Levinthal's paradox ?

Proteins fold much faster than it takes for then to sample all possible conformations.3. (40 points)

What does SDS stand for?

Sodium dodecylsulfate

Using X‑ray crystallography, Rosalind Franklin was able to produce X‑ray diffraction images of DNA that were clearer than any previously produced images. What did the images show regarding the structure of DNA?

The complementary strands of a DNA molecule form the shape of a double helix.

When sequence is treated with trypsin, the amino acids is cleaved. What do these results suggest regarding the sequence of the protein?

The protein has a lysine or arginine at its C‑terminal end.

The genetic code is said to be degenerate. This means that

an amino acid may be specified by more than one codon.

The process wherein two separated DNA strands reunite to form a double helix is called

annealing

What are negatively charged amino acids?

aspartate (D), glutamate (E)

Which of the following can SDS polyacrylamide electrophoresis be used to do? a) purify a monomeric enzyme in its active form b) determine the molecular weights of subunits of an oligomeric protein c) determine the molecular weight of an oligomeric (multisubunit) protein d) determine the folding state of a monomeric enzyme

b) determine the molecular weights of subunits of an oligomeric protein

What reagent did Anfinsen use in his protein folding experiments to reduce disulfide bonds in proteins?

beta-mercaptoethanol was used to reduce and break down the disulfide bonds holding the tertiary structure together

What is the bp recognition for HaeIII?

blunt GGCC

Which of the following is the LEAST useful for determining the three‑dimensional structure of a protein? a) x‑ray crystallography b) cryoelectron microscopy c) analytical ultracentrifugation d) nuclear magnetic resonance spectroscopy

c) analytical ultracentrifugation

What is rRNA?

combines with protein to form ribosomes

What are the generic names of the two steps that are repeated each cycle during solid phase peptide synthesis? The steps should be in sequential order.

coupling deprotection

How to calculate purification level on a protein purification experiment?

current specific activity/ original specific activity

What are the special cases amino acids?

cystine (C), glycine (G), proline (P)

If a solid line represents a covalent bond and a dotted line represents intermolecular attraction, which of the choices shows a hydrogen bond? a) H-H b) H4C----H-F c) H20-----H-CH3 d) H3N----H-O-H

d) H3N----H-O-H

Which of the following can SDS polyacrylamide electrophoresis be used to do?

determine the molecular weights of subunits of an oligomeric protein

What are hydrogen bond donors? acceptors?

donors= H acceptors= N,H

What type of amino acids are likely to be on the solvent‑exposed surface once the peptide folds into its native conformation?

electrically charged or polar uncharged amino acids -NOT hydrophobic molecules b/c water is here

Why do you think they are called sticky ends?

enzymes leave single stranded overhangs that are helpful in holding 2 DNA pieces together-able to stick to complementary singe stranded DNA

Do large protein elute first or last from a gel filtration column?

first

Which intermolecular force is responsible for base pairing in DNA?

hydrogen bonding

The major interaction stabilizing the double helical structure of DNA is

hydrogen bonds between bases on opposite DNA strands.

Water‑soluble proteins, such as myoglobin, tend to fold such that

hydrophobic amino acid R groups are on the interior of the protein and hydrophilic groups are on the outside.

For soluble cytoplasmic proteins, do hydrophobic amino acids prefer to be on the (inside or the outside) of the protein?

inside

One of the benefits of the degeneracy of the genetic code is that

it lessens the chances of harmful mutations.

Do larger or smaller proteins elute from a gel-filtration column first

larger

Which two amino acids appear identical when using mass spectrometry?

leucine (L) and isoleucine (I)

What are positively charged amino acids?

lysine (K) , arginine (R), histidine (H)

Many scientific methods are used to determine protein mass. Which of the following methods would yield the most precise estimate of protein mass?

mass spectrometry

Breaking disulfide bonds is a necessary step in preparing proteins for sequencing. Which of the following reagents is used to accomplish this?

mercaptoethanol

Given kDA, how do molecules elution from a gel filtration column in order of their kDA amount

most kDA elutes first

If the pI of a protein is less than the pH of the solution, the protein will have a net ______ charge.

negative

What is the charge (positive or negative) on cation exchange resin?

negative -b/c it has cross-linked polymer with negatively charged structural units

If a protein has a pI of 6, will it bind strongly to a cation exchange column at pH 8?

no

Does a protein with a pI of 6 stick to cation exchange resin if the buffer pH is 7 (yes or no)?

no bc/ pI is less than pH

Could a negative ΔH and positive ΔS ever result in an unfavorable reaction?

no because ΔG is positive so its always favorable

The difference between a nucleoside and a nucleotide is that

one contains phosphate attached via an ester linkage.

What kind of reaction is the conversion of cysteine to cystine?

oxidation

What does PAGE stand for?

polyacrylamide gel electrophoresis

If the pI of a protein is greater than the pH of the solution, the protein will have a net positive or negative charge?

positive

A disease in which the infections particle contains only a protein is called a _____________ disease.

prion

The use of phenyl isothiocyanate, the key reagent in the Edman degradation, makes it possible to

remove and identify one amino acid at a time.

What are amino acids with polar uncharged side chains?

serine, threonine, asparagine, glutamine

Is it easier for hydrophobic or hydrophilic molecules to pass through the phospholipid bilayer?

small molecules and larger hydrophobic molecules move through phospholipid bilayer easily.

___________ proteins move faster on an SDS-PAGE. Large or Small?

smaller proteins -The smaller proteins move faster because they can penetrate both large and small pores, while the large proteins can only penetrate the larger pores

What does carboxypeptidase tell?

tells arginine, proline, or lysine is last amino acid on C-terminal

DNA is more resistant to hydrolysis than RNA. What feature of DNA is responsible for this characteristic?

the absence of a 2′‑hydroxyl group in deoxyribose

Protein folding is often described as a highly cooperative process. This means that

the folding of a protein is largely an all‑or‑nothing process.

The major conclusion of the experiment of C. Anfinsen involving ribonuclease was that

the information on how a protein should fold is contained in its amino acid sequence.

When α helices and β sheets are described as being amphipathic, it means that

they have one side or face that is predominantly polar, with the other side being predominantly hydrophobic.

How to calculate specific activity on a protein purification experiment?

total activity (units)/ total protein (mg)

Suppose that you want to radioactively label DNA but not RNA in dividing and growing bacterial cells. Which radioactive molecule would you add to the culture medium

tritiated thymidine

In Anfinsen's classic protein folding experiments what reagent did he use to denature the protein?

urea

Can dimeric protein have quaternary structure (yes or no)?

yes

What are the delta H and delta S signs for a spontaneous reactions above a certain temperature?

Δ H= + Δ S= +

What are the delta H and delta S signs for a spontaneous reactions in reverse at all temperatures?

Δ H= + Δ S= -

What are the delta H and delta S signs for a spontaneous reactions at all temperatures?

Δ H= - ΔS= +

What are the delta H and delta S signs for a spontaneous reactions below a certain temperature?

Δ H= - ΔS= -

How to calculate delta G?

ΔG= Δ H- T(ΔS) -If negative, then favorable.