BIOC Slides 2 (Amino Acids + Proteins)

What are proteins and their characteristics?

• linear polymers built out of a.a

• a protein’s final 3D structure depends on its sequence of a.a

• proteins can interact with each other and other molecules to form complexes

  • ex. hemoglobin → combo of 4 different polypeptides

• Proteins can be rigid or flexible

How many amino acids are there in living things and how are they used to make different proteins?

• there are 20 key amino acids in living things

• you can vary the a.a by caring the side chains (R)

aliphatic

• a compound with an open chain structure (alkane)

What are the non-polar and aliphatic R groups for amino acids?

1. Glycine (Gly, G)

2. Alanine (Ala, A)

3. Valine (Val, V)

4. Methionine (Met, M)

5. Proline (Pro, P)

*all of these are also hydrophobic

Describe Glycine:

• What is the abb.?

• structure?

• features?

• Gly, G

Features:

• the simplest a.a, R group is H

• the only archiral a.a

• technically not really alipathic or hydrophobic (but closet catergory, so we put it here: non-polar and aliphatic)

Describe Alanine:

• What is the abb.?

• structure?

• features?

• Ala, a

Features:

• contains a methyl group

Describe Valine, Leucine, and isouecine:

• What is the abb.?

• structure?

• features?

• Valine: Val, V

• Leucine: Leu, L

• Isouecine: ile, I → i

Features:

• all contain hydrocarbon side chains (R)

• ile also has a second chiral carbon

Describe Methionine:

• What is the abb.?

• structure?

• features?

• Met, M

Features:

• also has a hydrocarbon side chain (R), expect it has a non-polar thio-ether (C-sC) group

• hydrophobic R group

Describe Proline:

• What is the abb.?

• structure?

• features?

• Pro, P

Features:

• has an aliphatic side chain with a twist

• end of the R groups is bound to the N of the amino group

• this forms a 5 membered ring

• it is not aromatic

• ring structure makes the a.a. more restrained

• introduces kinks into a.a chain (i.e polypeptides)

What do all the non-polar and aliphatic R groups for amino acids have in common?

• all of these amino acids are hydrophobic and tend to cluster together

• different sized and shaped R groups allow for close packing

• they are usually found in the center of a protein away from water

  • driven by the hydrophobic effect (ie. increased entropy when hydrophobic molecules cluster together)

• usually not reactive

Describe features of all aromatic R groups:

• contain aromatic ring (phenyl rings)

• participate in hydrophobic interactions

What are the amino acids with aromatic R group

• Phenylalanine

• Tyrosine

• Tryptophan

Describe Phenylalanine:

• What is the abb.?

• structure?

• features?

• Phe, P

Features:

• contains a hydrophobic phenyl ring

Describe Tryrosine:

• What is the abb.?

• structure?

• features?

• Try, Y

Features:

• similar to Phe, execpt it has a reactive polar OH group which can H-bond

Describe Tryptophan :

• What is the abb.?

• structure?

• features?

• Try, W

Features:

• contains an indole group

  • indole group: a 5 membered ring connected to a 6 membered group

• this is aromatic

• NH is reactive and can H bond

What are the positively charged R groups in amino acids?

• Lysine

• Argine

• Histidine

These are basic!

Describe lysine and arginine

• What is the abb.?

• structure?

• features?

lysine: Lys, K

arginine: Arg, R

Features:

• contain long chains with ionizable groups

• Lys = amino group

• Arg= guanidinium group

• they are both postively charged at pH (pka >/= 10)

Describe Hisitidine:

• What is the abb.?

• structure?

• features?

• His, H

Features:

• also has an ionizable group (imidazole ring) with a pka 6

• thus, pure His at pH 7 will have an unchanged imidazole ring

  • imidazole ring: a 5 membered ring with N on 1 and 3

• however His in protein will often have altered pka, closer to pH 7 and exist as a mixture of its acid (pos. charged) and c. base (unchanged forms)

• this makes is a good proton donor and proton acceptor

  • this means H can act as a buffer (acid-base catalyst)

• it can be charged or uncharged depending on its location

• often found in the active site of enzymes

What do all negatively charged R groups have in common? - amino acids

• contain carboxyl groups as R-groups

• negatively charged at pH 7 and pka is below 4

• acidic

What are the negatively charged R group amino acids?

• Aspartate

• Glutamate

Describe Aspartate and Glutamate:

• What is the abb.?

• structure?

• features?

• Aspartate: Asp, D

• Glutamate: Glu, E

Features:

• Glu is 1 carbon longer than Asp

• remember “-ate” at pH 7 but “-ic acid” is strong acid

• charged amino acids are often found on the surface of proteins where they interact with water away from the hydrophobic a.a

Describe what Polar R groups all have in common?

• uncharged R groups at pH 7

• can H bond, more hydrophilic

What are the amino acids with polar R groups?

• cystine

• asparagine

• glutamine

Describe cystine:

• What is the abb.?

• structure?

• features?

• Cys, C

Features:

• contains a sulfhdryl or thiol (SH) group

• is polar and weakly H bond is reactive

• can form disulfide (covalent) bonds

  • this can link 2 parts of a chain or 2 separate chains together

• done by the oxidation (loss of e-) of 2 cysteine residues to cystine residues (non-polar)

Describe Asparagine and Glutamine:

• What is the abb.?

• structure?

• features?

• Asparagine: Asn, N

• Glutamine: Gln, Q

Features:

• are detrivatives of asparate and glutamate

• contains a terminal carbonyl amide

• terminal amine is usually uncharged

What is a primary structure?

• linear sequence of amino acid linked by peptide bonds to form a polypeptide

what is a peptide bond?

• linkage of an alpha-carbonyl of one amino acid to the alpha amino group of another and the loss of H20.

• the formation of a peptide bond isn’t energetically favourable but once it is formed it is stable and a high activation energy would be need to break the peptide bond

What is a polypeptide?

• is a series of amino acid residues linked by a peptide bond

What is a residue?

• an amino acid unit in a polypeptide

What does it mean when you say polypeptides have polarity?

• one end has a free amino (NH3+) (left side)group and one has a free carboxyl group (COO-) (right side)

• A polypeptide consists of a backbone of repeats with variable side chains

• the backbone is polar (hydrophilic) and rich in H-bonding potential

• therefore all the carboxyls and amines can H bond with the exception of proline, which has limited H bonding ability.

how many amino acids residues are typically in a protein?

• the polypeptide chain can contain 50-20,000 amino acid residues

How is a protein’s molecular weight measured?

• Daltons or kilodalonts (kDa)

• 1Da = 1g/mole

What does the primary sequence of amino acids allow us to know?

1. Determine shape

2. Determine the function ex. catalyst function of enzyme

3. understand diease ex. cystic fibrosis

4. understand evoluntary history

Point mutation nomenclature

What is the structure of an alpha amino acid?

• is like the ultimate lego set

• contains an essential carbon (c-alpha) attached to:

  • an amino group

  • carboxyl group

  • hydrogen

  • unique side chain R

• note the c-alpha is chiral

what is a chiral center?

• an atom with its substituents arranged so the molecule is not superimposable on its mirror image

• for each amino acid there are 2 enantimors expect glycine

enantimor

• a pair of molecules with atleast 1 chiral center that are mirror images of each other

L and D configurations and which one is more common in nautre?

• L is more common in nature

Zwitterionic form

• ions with both pos and negative charge but over all net charge is zero.

Why are polypeptides conformally constrained?

• The peptide bond has a double bond characteristic

• this is because of resonance between the peptide and the carbonyl

• as a result the peptide bond is planar

• this is turn locks a series of atoms into a plane

• so there are not rotations about the peptide bonds

what orientations do double bonds exist in and how does that impact peptide bonds?

Double bonds exist as:

• cis

• trans

therefore:

• peptide bonds can only be cis or trans, since they act like double bonds

• however, because of steric hinderness, all peptide bonds are trans expect for x-pro peptide bond where both cis and trans occur

Why are peptide bonds flexible?

• the bond between N-C(alpha) and the bond between C alpha CO are free to rotate

• this provides flexability, allowing the backbone to fold in many ways

dihedral angle

• how we measure the amount of rotation about the bond

  • this ranges from -180 - + 180 degrees

• note: this is different from bond angle

  • bond angle: a bend in the bond between molecules

  • dihedral angle: how the molecules rotate/twist due to the bond

Phi and psi bonds?

how to find the N-C alpha angle?

the N-C alpha angle = Phi + psi

Can all combinations of phi and psi be formed? why or why not?

• no, not all combos are allowed due to steric hinderness

  • this further limits the number of structures a protein adopts

• The possible combos are shown in a Ramachandran plot

Describe a Ramachandran plot

• areas of dark colour = very favourable

• areas of light colour = less favourable, but possible

• white areas = not permitted

why are random coils formed?

• large molecules that can freely rotate among many bonds will assume random coils (i.e. a mix of different structures)

why do proteins fold into a single structure?

• because proteins have a series of limitations on what orientations they can adopt (i.e the planar peptide bond and limitations on dihedral angles) they can often spontaneously fold into a single structure.

Secondary structure of proteins

• is the spatial arrangement of amino acid residues in a polypeptide that are relatively close to each other in a linear sequence (alpha helices and beta sheets/strands)

alpha helix

• polypeptide backbone forms the inner part of a right-handed helix with the side chains sticking outwards

• the helix is stabilized by intra-chain H bonds between the NH and CO groups of the backbone

Characteristics of alpha helix?

1. the R groups in an alpha helix are almost perpendicular to the axis of a helix

2. it has dihedral angle of phi =-60 degrees and psi = - 45 degrees

3. the c= o of residue i forms H-bonds with the N-H of residue i+4 (4 residues further)

4. all the N-H and C=O in the backbone are H-bonded expect at the ends

5. each amino acid residue in the helix increases the helix length by 1.5 A (helix rises by 1.5A per a.a.)

6. the R-groups of i, i+1, and i+2 point in different idreactions

7. the R-groups of i, i+3 and i+4 point in similar direactions

8. the helix is almost always right handed in proteins

9. Left handed helices are permitted but rare (not as stable)

10. ALpha helices are shown as twisted ribbons or rods

What is the alpha helix content in proteins

• vaires

• some have no alpha helixes and other proteins are all alpha helics

• 2 alpha-helices can interwine into coiled colds

• usually alpha-helics are less than 45 A

is peptide formation favourable? why does peptide formation occur?

• peptide breakage is more favourable than formation

• but acitivtion energy makes it hard to break

What is a beta-sheet/strands?

• usually polypeptide strans from the same molecule

• associated as stacks or chains in an extended zigzag

• stabilized by interstrand H-bond between N-H and C=O groups

Describe anti-parallel B-sheet/strand structure

• fro each amino acid extends to B-streand by 3.5A (more spread out than alpha helix)

• the R-group of adjacent residues point in oppsite direactions perpendicular to the plane of the strand or sheet

• the strands are organized into sheets

• the N-Hand C=O of a single residue i on one B-strand H-bonds to a singla residue i on the other B-strand oppsite

• has dihedral angles of phi -139 degrees and psi +135 degrees

describe parallel B-sheet/strand structure

• like anti-parallel B-sheet expect B-strands run in same direaction

• each residue in the B-strand only extends the strand by 3.25 A

• have different dihedral angle phi = 119 degrees and psi +113 degrees

• the N-H of residue i in one B-strand H-bonds to residue i in the other B-strand

• C=O of residue i in the first strand H-bonds to N-H group of i+2 in the second strand

Describe general B-sheet and B-strands characteristics

• B-sheets can be mixed (i.e both parallel and anti-parallel B-stands)

• B-sheets cna be flat or twistied

• the distance between B-strands in primary strucutre a.a can be small or large

• B-strands can be large or small

• B-strand can be shown as borad arrows pointing to C-terminal end

• both can be built on itself to form a beta barrel

what are loops and turns

• non-repetitive secondary structure that connects and changes directions of regular repeating elements like alpha helix and B-sheets

• loops are generally longer and more flexible, and have less H-bonding

• wide range of phi and psi

• turns are shorter, tight, stretches stabilized by internal H-bonding

  • Prolines are common to give tight angles

  • Glycine accommodates angles incurred by proline

Tertiary structure

• the spatial arrangement of a.a. residues that are far apart from each other in linear sequence as well as the pattern of disulphide bonds

• the polypeptide’s 3D structure

• each protein’s tertiary structure is different

• is limited to a single polypeptide that is folded into a structure

• in most 3 structures, the dihedral angles for each residue fall into permissible areas (blue areas) of ramachran plot.

Myoglobin

• example of Tertiary structure

• oxygen storage protein in mammalian muscle

• single polypeptide chain of 153 aa residues

• contains a heme group (iron in protoporhyin ring) where the oxygen bind

• 70% of the chain is in alpha helices (total of 8 helices in protein)

• the rest are largley loops and turned

• the core of the protein is almost exclusively composed of hydrophobic residuces expect for 2 His residues which is needed by heme

• the surface is composed of molar polar/charged residues (some non-polar)

• myoglobin binds oxygen with high affinity and only releases it when the [O2] is really low

structural domains

• some proteins have multiple compact structures called domains linked by flexiable sections in the polypeptide these often have no defined sturcture

Quaternary structure

• the spatial arrangement of multiple folded structures (folded polpeptides) and the nature of their interactions along with disulfide bonds between subunits

• some proteins are composed with more than 1 folded polypeptide (subunit)

Homomer vs heteromers

• Homomer: subunits that are identical

• heteromers: subunits that are different

• multimer

• 2+ monoers combined

• some proteins must be monoers to survive

Protomer

• the base unit in a quat. structure (the basic structural subunit that repeats to form a larger protein complex)

• usually repeptive (but not always) monomeric (composed of a single unit) in nature

• But the structure is not the same as a monomer

Hemoglobin

• example of quat. structure

• oxygen transporter in mammals

• composed of 4 subunits (dimer of dimers)

• 2x alpha subunits (alpha-globin)

• 2x B-subunits (B-globin)

• Hemoglobin (Hb) cannot function unless it is a tetramer

• the protomer for Hb is an alpha-B-dimer

What drives protein folding?

• folding is driven by thermodynamics

  • finding the most stable complex, which is the most neg. delta G

  • the difference in free energy between folded and unfolded is a small difference

    • ~20-60KJ/mol

• mostly driven by entropy

  • the hydrophobic redsidues are excluded from water in the core whilst the hydrophobic residues are on the surface

How do we predict protein’s 3D structure

• AI programs like AlphaFOLD2 or RossetaFold predict protein structure based one primary structure

Why must the polypeptide have H-bonding?

• in order to bury the polar/hydrophobic bakbone of the polypeptide in the core it needs to H-bone

How can alpha helix structures get destablized?

• if unpaired, charged, or polar groups are in the hydrophobic core the alpha helix will destabilize

Are alpha helixes and B-strands hydrophobic, hydrophilic, or amphipathic?

• amphipathic

Can a portion of the primary seq. define secondary sequence?

• Yes and no

• certain a.a. residues are more likely or less likely to be found in and stabilized (or destabilized) alpha helices and B-strands/sheets

• experiments have shown that the exact same portion of a sequence in 2 different proteins can adopt different secondary structures

  • therefore, we can’t always determine secondary structure by looking at a portion of the primary sequence

• the overall tertiary structure is influenced by the secondary structure

Which amino acids are considered alpha helix wreckers?

• pro

• gly

• and to a lesser extent B-strand wreckers

What are 3 characteristics of protein folding?

1. folding tends to be an all or nothing process (usually either folded or not)

2. It is co-operative as one portion of the protein folds (eg. B-sheets)

   • it will influence how another portion forms

   • don’t need to sample every structure

   • there are usually many possible pathways so we depict this as a free energy funnel

3. In an unfolded protein state, there are many possible structures with high free energy, but as these species fold, the free energy decreases until you reach the folded state.

are all proteins a 3D structure?

• not all proteins have a 3D structure

• some proteins only fold into a single structure when they bind something

• some proteins are in equilibirum between 2 sturcutres

What are 3 methods to determine the 3D structure of proteins?

1. x-ray crystallography: uses x-rays to measure electron density (this was how structure of myoglobin and hemoglboin were determined)

2. Nuclear magnetic Resonance (NMR): measured the location of nuclei

3. Cryo EM: uses a beam of e- to visualize a frozen protein

What are the different types of post transitional modification?

1. Phosphorylation

2. Glycosylation

3. Hydroxylation

4. carboxylation

5. Acetylation

6. Methylation

Phosphorylation

• the attachment of phosphate group usually OH of an R-group (ser,Thr, Tyr)

• to activitate or inactive a protein

Glycosylation

• the attachment of 1 or more sugars to a residue (asn, Thr, Ser)

• common is surface lavelling most proteins on cell surface are glycosylated

Hydroxylation

• addition of OH group

• usually to Pro

• ex. fibre stabiliztion in collagen in skin, if someone has scurvy it could breakdown

Carboxylation

• add a carboxyl group

• usually to Glu

• important in clothing

Acetylation

• addition of an acetyl group to an amino group

• Lys, Arg

Methylation

• addition of a methyl group to an amino group

• Lys, Arg

• most proteins are cleaved or trimmed after synthesis

• this could activate or deactivtate them

  • ex. fibrogen gets cut into fibrin (active form)

    • important for blood clotting but don’t want clots all the time

• multiple proteins can be formed from a single long polypeptide

  • ex. virus

Protein folding as a funnel

• as you go down the funnel it is more stable

• sometimes proteins can get trapped because it takes alot of energy to unfold and refold into something more stable (makes chaperone proteins important)