MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 1 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
Introductory Comments: This is the first hour of the first of 3 Biochemistry lectures by Dr. Renfrow, and the first
transcript put together by the transcript committee so that you can see what to expect from the service. See Slides 2-4 for
Objectives.
I.
Proteins (slide 5) 3:45
a. Central Dogma, from DNA to Proteins
i. DNA is transcribed to mRNA which is then translated into a protein
1. A protein is a special polymer of amino acids with a lot of variety
2. This process involves a Ribosome, mRNA and tRNA attached to an amino acid (slide 6)
a. The tRNA brings amino acids in one at a time, where the ribosome makes sure everything is
oriented correctly before attaching the amino acid to the growing polymer chain to make a
protein. It is assembled one AA at a time.
b. The ribosome also proofreads while this is happening, although mistakes do still happen
during translation.
i. Polymer comes out in a “string fashion.”
b. Genesis of a Protein (slide 7) 5:17
i. The genesis of a protein involves more than just translation of mRNA
1. Sometimes, as mRNA is being transcribed, it can be put into the endoplasmic reticulum where the
protein will be modified almost immediately as it is coming out of the ribosome
2. After transcription, the final chain is linear so it must also fold into some kind of structure
a. Folding can happen all along the way, almost as soon as it gets out of the ribosome or later
on (such as when modifications occur), from 1°  2°  3°  4°  5°
b. Some parts may fold incorrectly or require “help” folding. This help comes from proteins
called “chaperones”
i. Chaperones make sure that the protein folds in the proper way
3. After folding you can then have post translational modification
a. The addition of different types of chemistry (e.g. sugars, phosphates, acetylation, methylation
etc.)
i. These modifications might tell the protein where it needs to go or what it needs to do.
b. You can also get modification of the actual side chains of the amino acids
c. Large Protein Assemblies (slide 8) 6:50
i. Example: RNA Polymerase 2 (SN: Dr. Renfrow worked with this in graduate school)
1. It is made up of 12 subunits with over 500 amino acids per individual subunit
2. They have to be assembled in the correct way
a. First you must translate each individual chain
b. Then those chains must fold and associate with one another in the correct way
c. Then chains and subunits will be modified in particular ways so that the final protein can fulfill
its particular task
i. In this case, RNA Polymerase 2 transcribes DNA to mRNA
d. Perspective (slide 9) 7:45
i. DNA and Genes are only what might happen
1. We have a copy of all of our genes in every cell, but not every gene is expressed at any one time
ii. mRNA is what is trying to happen
1. Just because it gets transcribed doesn’t mean it gets translated
2. Different factors and mutations may inhibit that process along the way
iii. Proteins are what is happening now
e. General Considerations for Proteins (slide 10) 8:40
i. The synthesis of a protein is energetically expensive
1. The degrees of freedom of individual amino acids are reduced
a. This restriction of motion costs energy because of loss of entropy.
ii. Products are fragile and unstable requiring constant proteostasis
1. Protostatis = “I’m happy to be folded like I am and doing the activity that I am, and if you mess with
that I may just fall out of solution, not work anymore, or work at 50% of my previous activity”
iii. Products are entrusted with crucial roles in the overwhelming majority of physiological functions
iv. All proteins have a molecular weight
v. All proteins have a molecular mass
1. Usually measured in Daltons (Da)
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 2 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
a. 220,000 Da = 220 kDa
2. You can even have proteins as simple as 500 Da
3. One mass unit = one twelfth the mass of a Carbon 12 atom
a. Everything else is referenced off of this mass
f. Protein Diversity (slide 11) 10:42
i. Different sizes
1. Example: Glucagon (29 AA’s, 3.2 kDa)
a. Glucagon is an antagonist for insulin
2. Metenkephalin (5 AA’s, 550 Da)
a. Metenkephalin is one of the originally discovered endorphins and the one that produces the
“runners high”
i. Endorphin is short for “endogenous morphine”
ii. There are several other endorphins, all of which differ from Metenkephalin by only one
amino acid
3. Collagen (1800 AA’s, 198 kDa)
a. Fibrous-type, connective tissue protein that is linked together in a series of coiled coils and
alpha helices. It allows us to make organs.
4. Titin (26,926 AA’s, 2,800,000 Da)
a. Largest known protein in existence
b. Made up of 244 individually folded protein domains connected by unstructured peptide
sequences
c. Involved in muscle tissue
ii. Different Shapes; Three Main Types
1. Fibrous
a. Example: Collagen
2. Membrane Bound
a. Example: Bacteriorhodopsin, also known as G-Protein coupled receptors
b. Membrane bound means they are in a phospholipid environment
i. Therefore, a good portion of the protein is hydrophobic
3. Globular
a. The majority of proteins mentioned in the literature because they stay in solution so you can
work with them
iii. All of these different proteins of different shapes, sizes and functions are all polymers of amino acids
assembled together in different ways and folded together in different ways so that they can function
iv. Different Functions
1. Catalysis – Enzymes (e.g. proteolysis)
2. Buffering – Serum Proteins
a. On a regular basis, our body stays at a pH of 7.4 and doesn’t like to deviate
b. In order to stay at this pH we need a buffering capacity
3. Storage – Collagen
4. Transport – Hemoglobin
a. Made up of 4 Heme groups (SN: Example picture on slide 14)
5. Motion – Actin-Myosin
a. Also Titin
6. Structure – Collagen
7. Defense – Antibodies
a. 2mg of IJ-1 from 5ml of serum; we produce gram amounts of IJ-1 every day
b. But must be folded and assembled correctly to work
8. Regulation – Hormones and Transcription Factors
9. Transducers – Receptors
a. Like the G-Coupled receptor example from slide 13
v. Interesting Examples
1. Albumin (66 kDa)
a. Highly anionic plasma protein (45 g/L) that maintains colloid osmotic pressure in our
circulatory system and transports minerals, fatty acids and drugs
b. See graph of distribution of all proteins in Serum at any given time on slide 15
i. Albumin takes up over half of the pie chart
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 3 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
A. So if you’re going to isolate something from the serum, you’ll always
have to account for albumin
2. Alpha 1-Antitrypsin
a. Inhibits serine proteases
3. p53
a. Controls the cell cycle and triggers apoptosis
b. As many as 50% of cancers have a mutation in p53
c. There are more mutations in p53 than there are amino acids
4. Scaffold Proteins
a. Promote cell assembly processes
i. Process of dividing a cell
b. Without a scaffold, you can’t divide because you can’t orient the organelles in the cell into the
right place so division can take place
5. Melanocortin 1 Receptor
a. Transmembrane protein that regulates pigmentation in vertebrate organisms
i. Transmembrane transport proteins allow salt in and out, allow things to break down and
leave, and allow different nutrients to come into the cell.
vi. Proteins have size, shape, location and function; but they are all polymer sequences of amino acids that
have chemical properties such as:
1. Affecting structure
a. Rigidity, flexibility, turning, order, disorder
2. Reactive
a. Produce active sites by bringing different parts of a protein together
3. Promote different interactions
a. Dipolar, hydrophobic, hydrophilic, hydrogen bonds
4. Hydrogen bonds are unique combinations that lead to
a. Specificity
i. Such as specificity of recognition
ii. Example: Loss of that recognition can lead to an autoimmune disease because you
now have an antibody that reacts to self
b. Specific shapes, predictable outcomes for reactions (once you know how things are
assembled), ability to transmit information, and can act as molecular switches
vii. Example: Transcription Factor
1. The ligand binding domain of a nuclear receptor (like estrogen receptor)
2. Structure, when bound to the ligand, activates transcription by binding DNA at the DNA binding
domain
II. Amino Acids: The Building Blocks of Proteins (slide 18) 20:00
a. Basics
i. There are 20 unique amino acids, which are used to make proteins
ii. Amino acids in proteins contain a central, tetrahedral carbon atom (except for glycine)
iii. Amino Acids polymerize and form proteins via peptide bonds
b. Structure of Amino Acids
i. Alpha Carbon in the center with R-Group, Amino group and Carboxyl group attached
ii. Usually viewed from N to C terminus
iii. Orientation
1. All of our amino acids are in the L confirmation, not D
2. As a result, all of our enzymes are assembled for L amino acids
a. Our enzymes do not recognize D amino acids
b. Some bacteria produce D amino acids as a defense mechanism in order to avoid being
recognized as antigens and broken down
iv. Amino acids have 20 different R-Groups
1. Most are in the ionized, zwitterion form at pH7
c. The Peptide Bond (slide 23) 22:00
i. Formation of the Peptide Bond
1. Involves a nucleophilic attack of the amino proton to the carboxylic acid
a. This leads to a loss of water and the formation of the peptide bond
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 4 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
b. All amino acids have positive and negative end, and the polymerization process takes
advantage of this when it sticks them together
2. The formation of a peptide bond eliminates:
a. Water (SN: Student Answer)
b. Rotation (SN: Student Answer)
c. Charge (SN: Answer Dr. Renfrow was looking for)
i. There is still a slight polarity, and it will be involves some in structural assembly
1. Oxygen has a slight negative charge because of the pull of the
carbon it’s attached to
2. Carbon Nitrogen bond always has a bit of a double bond character,
giving it a slight positive charge
ii. But they are no longer ionic as they were when they were separate
d. When you eliminate charge (a place where you can have reactions) the main players in the
polypeptide then become the R-Groups
i. Separate amino acids have functions by themselves
1. Example: Without Arginine you couldn’t produce nitric oxide and
dilate blood vessels quickly.
2. Example: Without Histidine you wouldn’t have Histamines and you
wouldn’t have allergy problems.
ii. But once you assemble them into a protein, you eliminate those charges and the side
chains become the major players
ii. Structure of a Peptide Bond
1. Planar – it fixes 6 (alpha carbon, carbonyl, anion, next alpha carbon) atoms into position
a. These atoms never move out of plane
i. Example: Think of them like hinges assembled together
2. Partial Double Bond
3. Partial Charge
4. Always Trans
a. R-Groups from each successive amino acid will end up on opposite sides
5. Limits degrees of freedom
6. Major decrease in Entropy
7. Movement can now only happen around Psi and Phi bonds
a. Alpha Carbon to Carbon bond is the Psi Bond
b. Alpha Carbon to Nitrogen bond is the Phi Bond
8. Series of peptide bonds become the “backbone” of the protein
9. Polymerized amino acids
a. Are a series of planes assembled together
i. Move like “hinges”
d. 20 R-Groups or Side Chains (slide 29) 28:04
i. Everything that the amino acids do while assembled into proteins are the result of the side chains
1. Affecting Structure
a. Rigidity, flexibility, turning, order, disorder
2. Reactive
a. Active sites, bring parts together
3. Promote Interactions
a. Dipolar, hydrophobic, hydrophilic, hydrogen bond, charge
4. Unique combinations that lead to
a. Specificity, specific shapes, predictable outcomes in reactions, transmit information,
molecular switches
ii. Types of Side Chains
1. Ten are Non-Polar Side Chains
a. Majority have carbon chains
b. Proline is unique because it is actually part of the peptide backbone
i. Makes it more rigid
ii. You can use Proline in specific spots to get specific geometries because of this
c. Aromatics
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 5 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
2. Five Uncharged Polar Side Chains
a. Two have amino groups, but they are not charged
b. Remaining three have polar hydroxyl group
3. Three Basic Side Chains
4. Two Acidic Side Chains
iii. T here are different ways to list out the types of side chains, some included on slides 30-32
1. Not one set way to group them
a. Example: Polar vs Non-Polar
b. Example: Aromatics in their own group
iv. All 20 have a set chemical formula
1. You can find the molecular mass, average occurrence in proteins, pK1, pK2 and pKr in the table on
slides 33-36
2. Tyrosine, Histidine, Lysine, Arginine, Cysteine, Aspartic Acid and Glutamic Acid all have side chains
that can ionize, and therefore have a pKr
v. Nomenclature
1. All 20 have a three letter code and a one letter code
a. See Slide 37 for list
b. Most often you’ll see the one letter code used
e. Types of Amino Acids (slide 38) 31:30
i. Basic Types
1. Ionic
2. Hydrophilic
3. Aliphatic
4. Aromatic
5. Sulfur
ii. Essential Amino Acids
1. Nine of the amino acids we cannot make ourselves, and therefore they have to be included in one’s
diet
a. These (found on slide 39) are:
i. Phenylalanine, Tryptophan, Histidine, Leucine, Isoleucine, Lysine, Threonine,
Methionine and Valine
iii. Non-Polar Amino Acids
1. Ten of the 20
2. Most have methyl groups at the end
3. Proline
a. Wants to be aromatic
4. Glycine
a. Only amino acid that is not chiral
b. Has a Hydrogen instead of an R-Group
c. Will never find a protein with long series of Glycines because they can’t have much effect on
structure
d. Glycine is a flexible filler for tight spaces
5. Two with sulfur groups on them
a. Methionine’s sulfur is occupied by a methyl group
b. Cysteine’s Sulfur is available through SH
6. Two non-polar aromatics
a. Phenylalanine and Tryptophan
i. Tryptophan does have a Nitrogen in it, but it is very hydrophobic and therefore never
charged
iv. Sulphur Groups
1. Methionine is non-polar and highly hydrophobic
2. Cysteine is very similar to Serine
a. Has SH rather than OH
b. Can form disulfide bonds
i. Very Important for protein structure
ii. Example: Antibodies could not form without disulfide bonds
1. First thing you do to analyze an antibody is to reduce disulfide bonds
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 6 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
2. Antibodies have two heavy chains and two light chains, but all along
the twisted parts of the antibody (see figure on slide 41) you have
disulfide bridges between adjacent Cysteines
a. Adjacent in space not linearly; Cysteines from one chain
associating with Cysteines from another
v. OH Groups
1. They are polar and hydrophilic but still uncharged
2. Have structural properties and are involved in hydrogen bonding
a. They have different scaffolds for H-bonding
i. Example: If you substitute a Serine with a Tyrosine in a particular protein you will create
a “bulge” because you’re taking up much more space
1. Even though it has the same functional group, it has a different
architecture upon which the OH group is assembled
vi. Nitrogen Containing Groups
1. Both have Amide side chains not Amine
2. They are polar, not charged at neutral pH, and are often involved in H-bonding
a. H-bonding comes into play with OH and Nitrogen containing groups and they are very
important in holding proteins together alongside other types of interactions
vii. Basic and Acidic Side Chains
1. Charged a majority of the time
a. Acidic side chains are usually negatively charged
b. Basic side chains are usually positively charged
III. Modification and Use of Amino Acids (slide 45) 37:07
a. Disulfide bonds between Cysteines
i. Loss of water forms disulfide bond
ii. Oxidation/Reduction reaction
iii. Very handy in assembling protein structures as well as breaking them down
b. Modification of OH groups
i. Modification with Sugars
1. Mucins on extracellular proteins are usually heavily modified
a. Can be attached to serine and threonine
b. Can attach GalNAc residues to Mucins in serum proteins
i. There are over 20 GalNAc transferases
1. Not all expressed at the same time
2. Can be in different tissues
ii. These serve to protect the protein from protease digestion
iii. They are also involved in cell-to-cell recognition
c. The amount of sugars decorating a protein can indicate whether or not the cell is acting right
d. Mucins can also be good markers for cancer
e. Example: Ij-1 with a region of 5-6 glycans from tail to head piece
i. This changes the usual “Y” shape of the antibody to a “T” shape, which is the shape of
Ij-1 most often found in your serum
1. This is also a result of a series of Serines, Threonines and Prolines
in that hinge region, which create this specific geometry to make it
bend out
ii. Modification with Phosphate Groups
1. Can be added to OH containing amino acids
2. Done using one of the 500+ Kinases available
a. Kinases add phosphates to proteins
b. Involved in signaling, conformational switches, activation and inactivation
3. Majority of Phosphorylation occurs on Serine
c. Modification of Nitrogen Containing Amino Acids
i. Asparagine is a site of N-Glycosylation
ii. Example: HIV related protein GP120
1. It is heavily N-Glycosylated (up to 24 sites)
2. This N-Glycoylation is changed depending on what cell it is expressed in
3. The majority of Anti-Virals made for HIV are made from a single cell population
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MS-1 FUND 1: 8:10-9:00
Monday, August 21, 2012
Dr. Matthew B. Renfrow, PhD
Amino Acids and Proteins
Transcriber: Dragos Rezeanu
Editor: Name
Page 7 of 7
Abbreviations: AA = Amino Acid, mRNA = messenger RNA, tRNA = transfer RNA
a. Because of this, the GP120 produced targets a specific level or type of glycosylation on the
HIV capsid
b. So if in the patient the HIV is expressed in a different type of cell, the drug won’t be as
effective
c. All this to say that it’s not just the amino acids that make a difference, but the additions and
modifications that occur after they’re assembled
d. Modification of Basic Amino Acids
i. Lysine can be monomethylated, dimethylated or trimethylated
ii. Can also add an acetyl moiety
1. Example: This kind of modification is common in histones
a. Histones package DNA too tightly to be transcribed
b. Require different combination of methyl and acetyl groups assembled on portions of the
histone to unwind the DNA
c. These and other modifications of side chains on histones are sometimes referred to as the
“Histone Code”
d. Histones assemble into four peptides.
e. The chemical modifications produce specificity.
e. Modification of Side Chains Themselves
i. Example: Proline  Hydroxyproline
1. Important in maintaining structure of Collagen
2. See other side chain modifications on slide 54
f. Schiff Base Formation
i. Reaction of a lysine side chain with an aldehyde
ii. In this way we reduce an unstable Schiff base into a more stable secondary amine.
1. Can be converted back to a primary amine when needed
No student questions
<END OF LECTURE 46:10>
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