Biochemistry - Intro to Proteins (Ch. 5) - Test 2

What is the sign for ΔG in peptide bond formation?

+ ΔG

What is the net charge on the following peptide at pH = 0? Peptide sequence: DSVK

+2

Choose two different ways you could change the buffer to elute the bound protein(s) and achieve proper separation of the proteins.

- add a buffer with pH = 8.0 - increasing the ionic strength of the buffer When the pH of the buffer is increased above 6.4, protein molecules become increasingly negative resulting in a decrease in the positive charge density on them. Thrombin, being less positively charged than cytochrome c, would elute first, followed by cytochrome c. Alternatively, increasing the ionic strength of the buffer would result in a competition between soluble ions in the mobile phase and charged residues of the bound proteins to bind the charged functional groups on CM-cellulose. Thrombin, being relatively less strongly bound to the CM-cellulose, would elute first, followed by cytochrome c.

What are some methods for hydrolyzing peptide bonds?

-boiling in strong mineral acid; very harsh and can destroy side chains -proteolytic enzymes (proteases); gentler because cleave only specific bonds

Which conformation, cis or trans, is least favorable based on the position of side chains of adjacent amino acids?

-cis is least favorable because it has the most steric hinderance -trans is more favorable because it spreads out R groups

How can an amino acid be calculated if it has ionizable R groups?

-identify the species of the amino acid that has a net zero charge by drawing out each ionic form of the amino acid, starting at low pH and removing the ionized hydrogen each time -average the two pKa values that surround the neutral species

Discuss why aspartame is an interesting peptide

-it is an artificial sweetener than the body can't metabolise -the amino acids are in the "L" configuration and are not metabolized by the body -if the amino acids are in the "D" configuration, they would not be metabolized, and the peptide is bitter rather than sweet in this configuration

Characteristics of conserved amino acid residues

-most likely located in the interior of the globular protein -might serve a critical structural or functional role in the protein -might be involved in ligand binding

Why are polypeptides excellent examples of polyampholytes?

-polypeptides usually contain some amino acids that have ionizable groups on their side chains -these groups have a wide range of pKa values, but are all weakly acidic or basic groups

How many nucleotide changes are required at the level of the gene for a lysine to asparagine mutation?

1 Lysine is coded by the codon triplets AAA and AAG, whereas asparagine is encoded by AAU and AAC. Hence, a single nucleotide change at the third nucleotide (AAA to AAU or AAG to AAC) is sufficient for a lysine to asparagine mutation to occur.

Human and sperm whale myoglobin have very similar primary structures. Which of the following statements are correct? 1) The two proteins are very likely related evolutionarily. 2) The differences in the sequences in many instances represent a conservative change (such as L for 3) The differences in the sequences in many instances represent a nonconservative change (such as D for A). 4)There is no correlation between the two proteins, since they originate from very different species.

1 & 2

Which of the following characteristics are true about a typical peptide (amide) bond? 1) The bond is planar. 2) There is free rotation about the carbonyl carbon and nitrogen bond. 3) There is substantial double-bond character to this bond. 4) There is a net negative charge on nitrogen and net positive charge on oxygen.

1 & 3

Which of the following characteristics are true about a typical peptide (amide) bond? 1.The bond is planar. 2.There is free rotation about the carbonyl carbon and nitrogen bond. 3.There is substantial double-bond character to this bond. 4.There is a net negative charge on nitrogen and net positive charge on oxygen.

1 & 3

To characterize the thrombin in the sample, you must remove two proteins that interfere with the thrombin activity assay: cytochrome c and lactoglobin. You find some CM-cellulose and a phosphate buffer (pH=6.4) on the shelf in your lab. You decide to load the protein sample onto a column of CM-cellulose equilibrated in the pH=6.4 buffer. Predict the order of elution for the three proteins shown in the table below. Protein................................pI Cytochrome c.................10.6 Lactoglobin......................5.2 Thrombin (wild type)....7.1 Rank from the first to the third.

1) lactoglobin 2) Thrombin (wild type) 3) Cytochrome c CM-cellulose is a cation exchange resin which carries a negative charge and binds to positively-charged groups. If the pH of a solution is less than the pI of a molecule, the molecule attains a positive charge density. Conversely, if the pH of the solution is greater than the pI of a molecule, the molecule will carry a negative charge density. In this case, pH of the buffer is 6.4, while the pI of cytochrome c is 10.6 which is greater than the pH of the solution. Hence, cytochrome c will have a positive charge density. Using the same reasoning, thrombin (wild type) has a pI of 7.1 which is higher than the pH of the solution, and thus will also carry a positive charge density. Conversely, lactoglobin will carry a negative charge density owing to its pI being lower than the pH of the solution, pH 6.4. It is pertinent to note that the pI of cytochrome c, which is 10.6, is much higher than the pI of thrombin (wild type), which is 7.1. As a result, cytochrome c carries a much higher positive charge density than thrombin (wild type). Hence, the order of elution will be: lactoglobin (which will flow through and not stick to the column), followed by thrombin (wild type) (which will elute slowly), and given time cytochrome c.

Most oligopeptides and polypeptides retain an unreacted amino groups at one end called _____1______, and an unreacted carboxylic acid group at the other end called ____2_____

1. amino terminus or N-terminus 2. carboxyl terminus or C-terminus

Neutral (neither acidic nor basic) amino acids can be further classified as nonpolar or polar. The neutral nonpolar amino acids have R groups that contain no charged atoms; most of these amino acids are _______1_________. The neutral polar amino acids have R groups that have a dipole moment. The partial charges in their R groups make these molecules _________2__________

1. not water soluble 2. generally water soluble

The average molecular weight of amino acids is 128. Each peptide bond removes a water molecule with a mass of 18. Thus, a crude estimate of molecular weight is to multiply the number of amino acids by ______.

110

While there's no specific cut off that defines a polypeptide from a protein, insulin is often described as the cutoff- around ______ residues.

51

Polypeptide

A polymer (chain) of more than 15-20 amino acids

Why are aspartic acid and glutamic acid usually referred to as their conjugate bases- aspartate and glutamate?

Because the side chain pKa values are so low that the negatively charged form of the side chain typically predominates under physiological conditions, even when incorporated into proteins

During ribosomal protein synthesis, amino acids are added to the ______________ end of the growing peptide

C-terminal

Molecular weight is typically reported in ________, which is essentially equivalent to g/mol.

Daltons (Da)

Name the N-terminal and C-terminal residues for the following peptide sequence. Peptide sequence: EASY

Glutamic acid is the N-terminal residue; tyrosine is the C-terminal residue.

What is the only achiral amino acid? It is found in flexible parts of proteins due to its tiny R group.

Glycine

Glycine, alanine, valine, leucine, isoleucine, and proline are considered amino acids with nonpolar aliphatic side chains. Which of these amino acids are often observed on the surface of protein?

Glycine and proline because of their ability to form tight turns in protein structures. Turns are sites where protein polymer folds back on itself also because they both have hydrophilic R groups?

Based on their side-chain structures, compare and contrast the potential of Lys and Asn to form noncovalent interactions. In other words, can each form H bonds and/or salt bridges and/or van der Waals contacts?

H bonds - Asn & Lys Salt bridges - Lys VDW - Asn & Lys

Why is the peptide bond planar?

It exhibits partial double-bond character, preventing rotation.

Which isomer is the only one known to be incorporated into proteins?

L-isomer

All amino acids except glycine are chiral, with the two isomer forms known as __________ and __________

L-isomer and D-isomer

Do you think Lys-to-Asn (or Asn-to-Lys) is likely to be a frequently observed mutation in proteins?

Lys-to-Asn (or Asn-to-Lys) is likely to be a frequently observed

What are the residues called at each end of an amino chain?

N-terminus and C-terminus

Can the peptide bond rotate?

No, double bond character prevents rotation

Many of the D-amino acids found in nature have been discovered in bacterially produced peptides that have antibiotic properties. Bacteria secrete these peptides into their environments to kill competitor bacteria and thereby gain a selective advantage. Given your answer to part (a) of this question, what potential advantages might D-amino acids confer to a secreted peptide toxin?

Peptides containing D-amino acids are less likely to be recognized as substrates by proteolytic enzymes in the gut.

What are Ramachandra plots?

Plots the allowed angles for ψ and Φ

Answer: pI = 1/2 (pK1 + pK2) 2.34 + 9.60/2 = 5.97

Question: Find the pI of glycine

What are the side chains

R groups of the amino acids

The melanocyte-stimulating peptide hormone α-melanotropin has the following sequence: Ser−Tyr−Ser−Met−Glu−His−Phe−Arg−Trp−Gly−Lys−Pro−Val Write the sequence using the one-letter abbreviations.

SYSMEHFRWGKPV

Answer; pKR is pKa for side chain pI = 7.59 average pKa values around structure that has zero charge

What is the pI of histidine?

You are surprised to observe that the patients thrombin flows through the CM-cellulose column at pH=6.4 and does not bind. Confident in your technique, you suspect the patients thrombin is different from wild-type thrombin. Using a different buffer system, you manage to purify some of the patients thrombin, and you submit the purified sample for amino acid sequencing. The sequence analysis shows that the patients thrombin contains a mutation in the enzyme active site. A lysine residue in the wild type has been mutated to an asparagine in the patients thrombin. Does this mutation explain the anomalous CM-cellulose binding behavior you observed?

Yes. Loss of a (+)-charged side chain will increase the (−) charge density on the protein, making it less likely to bind to the CM-cellulose.

How is a polymer formed with amino acids?

after a condensation reaction between the carboxylate group of one amino acid and the amino group of another amino acid

What amino acid contains a methyl side group and comprises almost 8% of the residues in proteins.

alanine

What amino acids have aliphatic side chains?

alanine, valine, leucine, isoleucine, glycine, methionine, proline

What are the neutral nonpolar hydrophobic amino acids?

alanine, valine, leucine, isoleucine, glycine, methionine, proline

What atoms of the peptide bond are coplanar?

all of them

Amino acids can be covalently linked together by the formation of ________ bond

amide

Carboxylic acids can form what three carboxylic acid derivatives?

amides, anhydrides, and esters

What groups can undergo hydrolysis?

amides, anhydrides, and esters

The proportion of each amino acid remaining in the chain during the process of ribosomal protein synthesis is called ____________

amino acid residue

Amino acids have ionizable groups, and thus have titration curves. What are the ionizable groups?

amino, carboxylic acid groups, side groups (few)

Amino acids, peptides, and proteins are all _________; each has an isoelectic point

ampholytes

Which amino acid contains a guanidinium group? The guanidinium group has a very high pKa of 12.5, so this side chain is almost always protonated in biological systems

arginine

Which amino acids are modified from the acidic side chain to have an amide group instead of a carboxyl group? The amide group does not ionize.

asparagine and glutamine

What are the negatively charged acidic amino acids?

aspartate (aspartic acid), glutamate (glutamic acid)

Which amino acids contain acidic side groups? These groups have low pKa values around 4 and are typically deprotonated in solution

aspartate and glutamate

What are the amino acids with negatively charged (acidic) side chains?

aspartic acid/aspartate, glutamic acid/glutamate

What is the general tertiary structure?

attractions between secondary structure elements -the way they all fold together

If an amino acid has no ionizable R group, the isoelectric point can be found by ______________

averaging the two pKa values

Why is the process of forming a peptide bond thermodynamically unfavorable in an aqueous environment?

because it requires the elimination of a water molecule

What biological functions are proteins the main agents for?

catalysis, transport, structure, and motion

What is the general primary structure?

chain of amino acids

Oligopeptides

chains of fewer than 10 or 15 amino acids

As a class of biomolecule, peptides have low membrane permeability (i.e., few peptides readily cross the membrane bilayer). This limits most peptide-based drugs to targets that are on the surfaces of cells (rather than in the cytoplasm or the nucleus). Review the information in the figures below and propose a reason that most peptides are not likely to cross the membrane bilayer. Peptides typically carry _____________ groups (i.e., N- or C- termini and some side chains) that reduce transport across the ____________ core of the membrane bilayer

charged groups, hydrophobic

The primary sequence of a protein is encoded in DNA triplets called _________

codons

The formation of esters and amides is a result of ______________ reactions, which result in the production of water

condensation

Two amino acids can form a dipeptide through a _______________ reaction that yields an amide peptide bond

condensation

Which amino acid is the only one that can form disulfide bridges?

cysteine

At pH=6.4, which protein(s) do you predict will remain bound to the column with minimal flow through CM-cellulose?

cytochrome c As explained in Part A, at pH 6.4 thrombin (wild type) would elute slowly from the column. While the pI of thrombin is 7.1, and above that of the buffer with a pH of 6.4, the relatively small difference between the two values lets one predict that it will likely elute, just slowly. While cytochrome c has a pI that is much higher than the pH of the eluting buffer, and can be predicted to remain bound to the column with minimal flow.

A molecule is ___________ when pH > pka

deprotonated

At high pH, amine becomes ___________. This yields an anion.

deprotonated

At neutral pH, carboxylic acid becomes _____________ and amine remains _________. This gives a neutral zwitterion.

deprotonated, protonated

Peptide hydrolysis is ___________, but has a very high activation energy.

exergonic

Acidic amino acids have acidic R groups. Their electrically charged R groups make these molecules ____________ in water.

highly soluble

Basic amino acids have basic R groups. Their electrically charged R groups make these molecules ________________ in water.

highly soluble

What amino acid contains an imidazole group? The imidazole group has pKa of 6 making it the side chain most likely to act like a buffer in biological systems

histidine

Which is the least basic amino acid with positively charged side chain?

histidine

What are the amino acids with positively charged (basic) side chains?

histidine, lysine, and arginine

What is the favored reaction for a peptide bond at room temp in aqueous solution?

hydrolysis of peptide bond, with equilibrium lying to the right

Favorable charge-charge interactions between amino acid side chains in the protein hemoglobin result in ___________ oxygen delivery to respiring tissues

increased -Bohr effect

Estimate the molecular weight of insulin and reverse transcriptase (986 amino acids)

insulin = 51 amino acids 51 x 110 Da/ 1 aa = 5600 Da = 5.6 kDa transcriptase = 986 amino acids 986 aa x 110 Da/ 1 aa = 108.5 kDa

Insulin is a peptide therapeutic used to manage Type 1 diabetes, which affects more than 20 million people worldwide according to the International Diabetes Federation. A significant limitation to the broad distribution and use of insulin to treat Type 1 diabetes is the fact that it must be administered by injection rather than orally. Why is insulin administered by injection and not orally?

insulin would not survive transit through the stomach and gut environments

N-terminal residues is always written to the ____ and C-terminal residue to the ____

left, right

What are proteins?

long chains of amino acids that have functions

Which amino acid contains an amine group at the end of the long chain of 4-CH2 groups? This amine group often participates in hydrogen bonding and acid-base reaction

lysine

_________ and __________ side groups are almost always positively charged under physiological conditions

lysine, arginine

What are the positively charged basic amino acids?

lysine, arginine, histidine

Which amino acids contain sulfur?

methionine and cysteine

Aspartic acid and glutamic acid typicall carry _____________ charges at pH 7

negative

What is a zwitterion?

neutral molecule with positive and negative charge that cancel each other out

APFLLIGDWY is chain of amino acids, which can also be referred to as a(n)...

oligopeptide

What yields a disulfide bond?

oxidation of two cysteine side chains

glycine acid-base titration

pKa1 = carboxylic acid pKa2 = amine group cation- pH<pK1 zwitterion- pK1<pH<pK2 anion- pH>pK2 two buffering zones -> around two pka's pK1 = 1.34-3.34 (carboxylic acid buffering zone) pK2 = 8.6-10.6 (amine buffering zone)

What is another name used for amide bond

peptide bond, because these linkages form peptides

What is the reverse reaction for peptide bond formation?

peptide hydrolysis

What are the amino acids with nonpolar aromatic side chains?

phenylalanine, tyrosine, tryptophan

What are the aromatic amino acids?

phenylalanine, tyrosine, tryptophan

What are the neutral nonpolar aromatic amino acids?

phenylalanine, tyrosine, tryptophan

Some primary sequences must be _______________ to become active

post-translationally modified

What are the four levels of protein structures?

primary, secondary, tertiary, quaternary

What is the only secondary amino acid? The locked, cyclic structure of the side chain affects protein folding, and this amino acid is commonly found at bends in protein structures

proline

What is the general quaternary structure?

protein consisting of more than one amino acid chain -dimers, trimers, tetramers, etc

Polypeptides greater than ~50 residues are referred to as __________

proteins

What is the difference between a polypeptide chain and protein?

proteins have higher order structures, they are larger, and have a structure, function relationship

A molecule is __________ when pH < pka

protonated

At low pH (acidic pH), carboxylic acid and amine groups are fully _________. This gives a positively charged cation

protonated

Acid-base titrations show the addition and removal of _________

protons

Modification of the N- and C-termini (by, respectively, acetylation and amidation) will _________ the charge density on peptides. This would make them _______ likely to cross membranes.

reduce, more

What type of rotation around bonds is permitted?

rotations around bonds connected to the alpha carbon

What is the general secondary structure?

sequence of amino acids are linked by H-bonds -local with folding

What are the amino acids with polar side chains?

serine, threonine, cysteine, asparagine, glutamine

What are the polar, neutral amino acids? They are soluble in aqueuous solutions and often located outsides of protiens

serine, threonine, cysteine, asparagine, glutamine

What are peptides?

short chains of amino acids

Is the peptide bond stronger or weaker than a single bond?

stronger because bond order is 1.5

What does the primary structure of amino acids dictate in a protein?

structure and function, and folding

What is the amide bond between?

the alpha carboxylic acid group on one amino acid and the alpha amino group on another

For an amino acid such as aspartic acid, what impact would you expect a neighboring carboxylic acid to have on the apparent pKa value of the side chain?

the apparent pKa value for aspartic acid increases

Isoelectric point (isoelectric pH, or pI)

the characteristic pH at which the net electric charge on a species is zero

What does microenvironment take into account?

the chemical properties of a side chains immediate neighbors and can change the polarity of the side chain

What is the main chain?

the peptide backbone composed of atoms that make up peptide bonds, namely the N, C-alpha, carbonyl groups of each amino acid residue in the peptide -N-terminal amino group and C-terminal carboxylate are also part of the main chain

How are cis and trans conformations made across the peptide bond?

the single bonds of C-N and C-C can rotate

If polypeptides are thermodynamically unstable (like polynucleotides), then how can they be synthesized in the aqueous medium of the cell?

the unfavorable synthetic reaction is coupled to the favorable hydrolysis of certain organophosphate compounds -every amino acid must be activated by an ATP driven reaction before it can be incorporated into proteins

What is the charge on a protein in aqueuous solution that has a pH equal to the isoelectric point of the protien?

there is no net charge on the protein

Why are the amino acids with positively charged (basic) side chains usually found on exterior surface of proteins?

they are strongly polar, and can be hydrated by surrounding aqueous environment of exterior -they can also be found in substrate binding clefts of enzymes where they can interact with polar groups on substrate that binds to the enzyme

How are carboxylic acids affected by neighboring functional groups?

they generally have a pKa close to 4, but the presence of positive charge on neighboring group will cause the acidity of the carboxylic acid to increase

Why are peptide bonds metastable?

they hydrolyze rapidly only under extreme conditions or when catalysts are present

What is the geometry and the approximate angle for O-C-N?

trigonal planar, 120 degrees

Which aromatic amino acid contain an indole group and absorbs UV light strongly at 280 nm, which makes it useful for experimental detection of proteins. It is the least abundant amino acid in human proteins (1.4%)

tryptophan

What is a dipeptide

two amino acids joined by a peptide bond -involves elimination of a water molecule

Which amino acids contain a hydroxyl group? These amino acids are often found on the exterior of proteins, exposed to water because the hydroxyl group forms hydrogen bonds with water molecules

tyrosine, serine, threonine

What are the branch chain amino acids? These are the most hydrophobic amino acids and are usually found on the interior of proteins, away from water. These three amino acids are also essential nutrients

valine, leucine, isoleucine

A nonpolar amino acid at pH=7.0 will have a net charge of ______

zero at pH=7 an amino acid exists as a zwitterion (has overall charge of zero) it is polar due to positive charge on amino group and negative charge on carboxylic group, which cancel each other out

What are the types of rotations that can occur with the alpha carbon?

Φ (phi) - angle around alpha carbon-amide nitrogen bond Ψ (psi) - angle around alpha carbon-carbonyl carbon bond In a fully extended polypeptide, both Φ and Ψ are 180 degrees