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What are the most abundant biological macromolecules?
Proteins
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Where are proteins found?
- In all cells
- Even in viruses
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How many different kinds of proteins exist in a single cell?
Thousands of different kinds
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What are the different functions of proteins?
- Catalysis
- Transport
- Structure
- Motion
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What are the building blocks of proteins?
Amino acids
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How are amino acids organized in proteins?
Linear heteropolymers of alpha-amino acids
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How are amino acids bound to each other in proteins?
Through covalent peptide bonds
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About how many amino acids are known?
About 500
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How many different amino acids are commonly found in proteins?
20
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What amino acid properties are well-suited to carry out a variety of biological functions?
- Capacity to polymerize
- Useful acid-base properties
- Varied physical properties
- Varied chemical functionality
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Where do most amino acids differ?
The R (residue) substituent
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What is the center carbon in an amino acid called?
alpha carbon
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What is the general structure of an alpha amino acid?
- Alpha carbon in middle
- An acidic carboxyl group
- A basic amino group
- An alpha hydrogen connected to the alpha carbon
- R substituent
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What is the exception to the general alpha amino acid structure?
Proline
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How is proline different from other amino acids?
It's cyclic
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What are the two different ways to name amino acids?
- Organic nomenclature
- Biochemical designation
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How are amino acids named with organic nomenclature?
- Start from one end (carboxyl group) and go down
- Naming by numbers
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How are amino acids named with a biochemical designation?
- Start from the alpha carbon and go down the R group
- Naming by greek letters
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How many substituents does the alpha carbon in an amino acid have?
4
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Most alpha amino acids are ______, meaning they have __ different substituents.
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What is the fourth substituent in glycine?
Hydrogen
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What is unique about glycine?
It's the only non-chiral amino acid
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Proteins contain only what kind of amino acids?
L-amino acids
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Which formulas are preferred when studying chiral amino acid structure?
Projection formulas (Fischer)
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Why do proteins only contain L amino acids?
Our enzymes only recognize L amino acids
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What does L amino acid mean?
Amino group is on the left when looking at projections
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When are D peptides used?
- Many bacteria incorporate D-amino acids to their cell walls, to make them more resistant to peptidases
- Also components of many bacterially produced antibiotics
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How is the synthesis of D peptides different?
- Doesn't follow traditional DNA → RNA → Protein
- Instead, uses specific enzymes
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What is unique about cysteine?
Can form disulfide bonds
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What are the 5 groups that amino acids are classified into?
- Non-polar, aliphatic
- Aromatic
- Polar, uncharged
- Positively charged
- Negatively charged
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How many non-polar, aliphatic amino acids are there?
7
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How many aromatic amino acids are there?
3
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How many polar, uncharged amino acids are there?
5
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How many postively charged amino acids are there?
3
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How many negatively charged amino acids are there?
2
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What are the non-polar, aliphatic amino acids?
- Glycine
- Alanine
- Proline
- Valine
- Leucine
- Isoleucine
- Methionine
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What are the aromatic amino acids?
- Tyrosine
- Tryptophan
- Phenylalanine
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Which of the aromatic amino acids is the least polar?
Phenylalanine
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Which of the aromatic amino acids can form H bonds?
Tyrosine
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Which of the aromatic amino acids is the most aromatic???
Tryptophan
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What are the characteristics of aromatic R groups of amino acids?
- Hydrophobic
- Side chains absorb UV light at 270-280 nm
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How does the fact that the aromatic amino acid side chains can absorb UV light help with anything?
Can quantify proteins
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List the aromatic amino acids in terms of increasing max absorbance.
- Phenylalanine
- Tyrosine
- Tryptophan
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The aromatic amino acids absorb light in which light region?
UV region
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What is the typical UV absorbance maxima for proteins?
275-280 nm
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Which aromatic amino acids are the strongest chromophores (light absorber)?
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How can the concentration of an amino acid be determined?
By UV-visible spectrophotometry using Beers Law
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What is Beers Law?
- Used to quantify amount of protein/amino acid in a mixture
- A = ecl
- e = extinction coefficient (depends on compound)
- c = concentration of the absorbing species
- l = path lenght
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What are the amino acids with polar, uncharged R groups?
- Serine
- Threonine
- Cysteine
- Asparagine
- Glutamine
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What do the structural formulas of amino acids with polar, uncharged R groups show?
State of ionization that would predominate at pH 7.0
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What kind of bonds can be formed from polar, uncharged amino acid side chains?
Hydrogen bonds
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Name the amino acids that are amides of other amino acids.
- Asparagine - Aspartic acid
- Glutamine - Glutamic acid
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What happens when Cysteine oxidizes?
Forms covalently linked dimeric amino acid Cystine
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What is Cystine?
- Covalently linked dimeric amino acid
- Formed when cysteine oxidizes
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Where do the disulfide bonds in cystine usually form?
- In the endomembrane of systems or extracellularly
- Thiol group can't oxidize in the cytosol
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Why can't the thiol group of cystein oxidize in cytosol?
There are many reducing species available there
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How do disulfide bonds affect protein structure?
Stabilizes them
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Why is the formation of cystine important?
Important for determining structure of protein
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How do disulfide bonds stablize a protein?
- Holds two portions of the protein together, biasing the protein toward teh folded topology
- May form the nucleus of a hydrophobic core of the folded protein
- Can link different polypeptide chains
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What's an example of a disulfide bond forming the nucleus of a hydrophobic core of a folded protein?
Local hydrophobic residues condensing around the disulfide bond and onto each other through hydrophobic interactions
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What's an example of different polypeptide chains being held together by disulfide bonds?
Immunoglobulin heavy and light chains held together by DS bonds
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What are the amino acids with positively charged R groups?
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What's special about histidine?
- Only amino acid whose side chain pKa is relevant to us
- R group pKa of 6
- Works well as a physiological buffer near neutral pH
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What are the amino acids with negatively charged R groups?
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What are the amino acids with the negatively charged R groups called when they are protonated?
- Aspartate - Aspartic acid
- Glutamate - Glutamic acid
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What are the uncommon amino acids in proteins?
- Serine
- Cysteine
- Selenocysteine
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Why are the uncommon amino acids uncommon?
- Not incorporated by ribosomes (except selenocysteine)
- Arise by post-translational (covalent?) modification of proteins
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What kind of modificaitons of proteins are important in regulation and signaling?
- Reversible modifications
- Especially phosphorylation
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What is the origin for the single letter code for amino acids?
- Computers were primitive
- Dr. Margaret Dayhoff shortened the code to reduce data file size
- Some named by first letter, others phonetically
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How are peptides formed?
- They're condensation products of amino acids
- Extract water (OH + H) to bring amino acids together
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What are peptides?
A few joined amino acids
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How big are peptides?
Small compared to proteins
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Describe the structure of peptides
- Pretty linear
- Amino-terminal and carboxyl-terminal ends
- Carbonyl and amino groups are always alternating up and down
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How are peptides named?
- Start at N-terminus
- Can use full amino acid names, 3-letter codes, or one-letter codes
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What are the different functions of peptides?
- Hormones and pheromones
- Neuropeptides
- Antibiotics
- Protection, eg. toxins
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What are some examples of hormone and pheromone peptides?
- Insulin (sugar)
- Oxytocin (childbirth)
- Sex-peptide (fruitfly mating)
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What's an example of a neuropeptide?
Substance P (pain mediator)
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What are some examples of antibiotic peptides?
- Polymyxin B (for Gram - bacteria)
- Bacitracin (for Gram + bacteria)
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What are some examples of peptides as toxins/protection?
- Amanitin (mushrooms)
- Conotoxin (cone snails)
- Chlorotoxin (scorpions)
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Describe the ionization of amino acids at acidic pH.
- Carboxyl group is protonated
- Amino acid is in cationic form
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Describe the ionization of amino acids at neutral pH.
- Carboxyl group is deprotonated
- Amino group is protonated
- Net charge is 0
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Describe the ionization of amino acids at alkaline pH.
- Amino group is neutral (NH2)
- Carboxyl group is in anionic form
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What is a zwitterion?
A substance with positive and negative charges that neutralize so the net charge is zero
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When can amino acids act as buffers?
- Amino acids with uncharged side chains have two pKa values
- One for carboxyl group and one for amino group
- Can act as buffer in two regimes
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What are the 3 pKa values you should know?
- Carboxyl group: 2.34
- Amino group: 9.6
- Histidine side chain: 6
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What is the isoelectric point?
- pI
- Point of amino acid where net charge 0
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What can affect pKa values?
Chemical environment
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What is the inductive effect?
- Electronegative effect
- The more electronegative group pulls on the electrons of the amino or carboxyl group through single bonds
- This makes them more stable without the proton, giving them a slightly lower pKa
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How do the carboxyl and amino groups in an amino acid affect each other compared to their individual acid or base form?
- alpha-carboxyl group is much more acidic than carboxylic acid
- alpha-amino group is slightly less basic than in amines
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What are the acid-base properties of non-ionizable amino acids?
- Non-ionizable acids have titration curves similar to that of glycine
- pKa values of the carboxylic and amino groups are also similar.
- Any differences reflect the chemical environment imposed by the R groups
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At what pH do amino acids carry a net charge of zero?
- At the pI
- Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups
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What is the pI for an amino acid without ionizable side chains?
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When is an amino acid least soluble?
At the pI
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When does an amino acid not migrate in electric field?
At the pI
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What happens to an amino acid when the net charge is zero (pI)?
- AA is least soluble in water
- AA doesn't migrate in electric field b/c it's neutral
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Describe amino acids with ionizable side chains
- Ionizable side chains can also be titrated, but the curves are just more complex
- They have 3 pKa values
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How do you estimate the pI when the side chain is ionizable?
- Find net charge of amino acid at different pH values between the pKa's
- Find the species that carries a net zero charge
- Identify pKa value that defines the acid strength of this zwitterion (pK1; one pKa below the zwitterion)
- Identify pKa value that defines the base strength of this zwitterion (pK2; one pKa above the zwitterion)
- Take average of these two pKa values
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When are pKa values discernible for animo acids with ionizable side chains?
If two pKa values are more than 2 pH units apart
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Describe the backbone (and the ionization) of peptides
- Contain only one free alpha-amino group and one free alpha-carboxyl group at opposite ends
- They will ionize but the ionization constant is different from when it's just the amino acid b/c there isn't a charged group opposing them
- alpha-amino and alpha-carboxyl groups involved in peptide BONDS can't ionize
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What happens to the inductive effect of the N-terminal and C-terminal on a peptide?
- pKa values are a little different than in individual amino acids
- Reduced inductive effect because terminals are farther away from each other
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Describe the sidechains (and their ionization) of peptides
- The R group of some amino acids can ionize and contribute to the acid-base behavior of the peptide
- To note: the pKa for an R group can change when the AA becomes a residue in the peptide
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How do you find the pI of a peptide with ionizable side chains?
- Find zwitterion (net charge 0)
- Find two pKa's around it
- Take avg and get pI
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When are proteins the least soluble?
At their pI
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When do most proteins not have pI?
At cytoplasmic pH
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What can be used to experimentally separate proteins that have similar molecular weights?
Its pI
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When do pI of proteins vary?
If a protein has been coavlently bonded with a charged group
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Give an example of when the pI of a protein changed due to covalent bonds with a charged group
Protein will have different pI if phosphorylated
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What are carboxyl groups like at lower pKa values?
- The lower the pKa, the more acidic the environment has to be to add a proton
- B/c it's easier to lose protons
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What must happen to the carboxyl group in an amino acid for it to be easier to lose a proton?
Lower pKa
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What must happen to the amino group in an amino acid to make it harder for it to shed a proton?
pKa should be higher
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What is the difference between the alpha-carboxyl group of a glycine (amino acid) compared to that of a carboxylic acid?
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What is the difference between the alpha-amino group of a glycine (amino acid) compared to that of a amine?
- Slightly less basic/more acidic
- Lower pKa
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Why is the pKa lower for alpha carboxyl and amino groups in amino acids than those in carboxylic acids and amines?
- Electron withdrawal effect (Induction effect)
- Zwitterion is stabilized by the opposing charges (Zwitterion effect)
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How does the inductive effect affect the amino group of an amino acid?
- The electronegative oxygen in the carboxyl group makes the N more positive than it would be in an amine
- Easier to give up the H+
- Lowers the pKa
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How does the inductive effect affect the carboxyl group of an amino acid?
- The slightly electronegative nitrogen in the amino group makes the O slightly more stable as a negative (b/c pulling electrons) than it would be in a carboxylic acid
- Easier to give up the H+
- Lowers the pKa
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How does the zwitterion effect affect the carboxyl group of an amino acid?
- The positively charged amino group makes it so that there is repulsion between the H+ of the carboxyl group and the amino group
- Makes it easier to remove the carboxyl's proton
- Lowers the pKa
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How does the zwitterion effect affect the amino group of an amino acid?
- ??? Loook upppp
- Increases the pKa
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What has a greater effect on the amino group?
Inductive effect
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What is the additional resonance effect in peptide bonds?
- Carbonyl and amino group make a double bond
- This affects pKa of nitrogen
- Doesn't affect carboxyl at all
- b/c of resonance, electrons are a little less localized
- Nitrogen can end up having more positive density
- Lowers the pKa b/c proton is easier to get rid of
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