ch. 3 Protein structure

what are peptides and proteins to amino acids?

Peptides and proteins are polymers of amino acid

they are called polypeptides

What is a peptide bond? How is it formed?

A name given to the amide bond that joins two amino acids
peptide (amide) bond can be formed by condenstion

What type of molecule is a protein

Proteins are linear molecules of amino acids joined by amide (peptide) bonds formed by ‘simple’ removal of H2O

ex. aspartame

When looking protien backbone, describe the features

Repeating backbone is non-ionizable (but polar) exept the ends

Side-chains vary depending on amino acic

pKa, pI of side chains and termini are similar to free amino acid

Backbone has no charge and can form H-bonds

all polypeptides at the beginning has free amino and carboxyl

Backbone of a protein has an amino-terminus, internal ‘residues’, side chains, and carboxyl-terminus

What happens when peptide bonds are formed

alpha NH3+ and alpha COO- groups become covalently linked and can no longer ionize in solution

What are charged groups of polypeptides limited to

Limited to the two ends (termini) and some of the R groups

What are amino acid units in a peptide or protein called

Amino acid units in peptide or protein is called Residues

How are the amino acid sequence of peptides and proteins written

Written with the N-terminal residue on the left and the C-terminal residue on the right
NH3+-aa1-aa2-aa3-COO-

What are some characterisitics of polypeptide chains

Polypeptide chains are flexible (but still have constraints), so the way they fold up to form a compact, 3D structure is an essential aspect of protein structure

How is the folding of a protein determined

Folding of a protein is determined simply by its amino acid sequence. We can predict this as we have many protein structures (determined by x-ray crystallography, or sometimes NMR) and the basic principles of portein folding are understood

what are the four levels of protein structure?

Primary structure

Secondary structure

tertiary structure

quaternary structure

Describe the primary structure

• Linear sequence of amino acid residues

• The sequence/order of amino acids in the protein from terminus - terminus

• First protein seuence determined by Fred Sanger (1958, Nobel prize)

  • From the success followed by direct sequencing of thousands of proteins

  • Then he developed a method for DNA sequencing (1980 nobel prize)

• Most protein sequences determined from the DNA sequence of the corresponding gene by translation using the genetic code

What does Sanger protein sequencing involves?

1. Hydrolysis of the polypeptide (“digestion”) into small fragments, using specific cleavagte methods

2. Separating the small fragments and determining the AA sequence

3. Alignment of the small sequences to generate the complete sequence

Describe Fragmentation

For proteins, sequence specific proteinases (enzymes that cleave other proteins) and a few chemical reactions are used:

• Trypsin

  • cleaves after Lys or Arg (basic residues, positively charged)

  • All fragments will end with Arg or Lys except the C-terminal one

• Chymotrypsin

  • cleaves after Phe, Tyr, or Tryp (aromatic residues)

  • All fragments will END with Phe, Tyr, or Trp except the C-terminal fragment

• CNBr (Cyanogen bromide)

  • cleaves after Met residues

Describe Separation and Sequencing, Edman degradation

1. Edman degradation is used to determine the sequence of amino acids in a peptide, starting from the N-terminal (first) amino acid.

2. The core reagent is phenyl isothiocyanate (PITC). PITC reacts specifically with the N-terminal amino group of a peptide.

3. Under alkaline conditions, PITC reacts with the N-terminal to form a phenylthiocarbamoyl (PTC) derivative.

4. When this intermediate is treated with acidic conditions, the labeled amino acid is cleaved from the rest of the peptide as a cyclic compound called a phenylthiohydantoin (PTH) derivative.

5. The PTH-amino acid is identified using techniques such as chromatography. This identifies which amino acid was originally at the N-terminus.

6. The shortened peptide is subjected to another cycle to identify the next amino acid. This is repeated to sequence the peptide step by step.

• Sequence is read off one residue at a time up to 30 residues in favourable cases

What are limitations of edman degradation

• Works best for peptides up to 30–50 amino acids.

• Requires a free (unmodified) N-terminal.

• Less effective with blocked or modified N-termini.

Why would Mass spectrometry be better than edman degradation

while Edman is methodical, specific, and common, mass spectrometry is faster and more suitable for complex mixtures or longer proteins.

Describe what mass spectrometry for Primary sequence ID

Mass spectrometry is a technique in which the molecular masses of the different components in a mixture are measured with high precision and accuracy

What is the standard approach for mass spec.

first digest protein with a protease (like trypsin) and then identify the peptides based on their mass

1. Generation of ions

2. acceleration of ions in electric or magnetic field

3. separation of ions in mass Analyzer

4. detection of ions

Describe the process when using peptides with mass spec.

Peptides are ionized and sparyed into a vacuum chambber

bigger ions (larger mass) will fly more slowly and reach the detector later. Known as “time of flight”

‘Essentially a plot of intensity vs. mass

Describe the secondary structure

• regular conformational patterns of contiguous portionos of the polypeptide chain, stabilized by hydrogen bonds

• How parts of the sequence folds to maximize H-bonding between C=0 and NH3

• Local patterns and H-bonding

• There are two main types of secondary structure: alpha helix and Beta confromation (beta sheets)

What is an alpha helix?

Initially defined by Linus pauling - essentially an extended right handed spiral

Carboxyl is H-bonded to NH 4 places ahead

What are some determining features of the alpha helix

1. The peptide bond has ‘planar’ or double bond character through resonance structures to stablize the peptide bonds.
   The C-N peptide bond is shorter and rotation is constrained.
   The double bond character makes the peptide bond rigid and planar (peptide plane).
   Rotation possible around the alpha carbon but not around the O=C-NH bond.

2. R groups are in a ‘trans configuration’ (not a true double bond)
   The trans configuration of the alpha carbons as compared to the sterically less favourable ‘cis’ conformation.
   R groups clash in cis

3. Hydrogen bonds form between every 4th amino acid. The result of the partial double bond character is a partial postive charge on N and a partial negative charge on the O

4. R-groups tend to point away from the heliz, minimizing their contact; however, high concentrations of charged, bulky or small residues can disrupt the helix

5. alpha helix has a significant dipole moment.
   Affects: How helices cluster in a protein, bias of charged amino acids to particular end, and interaction with membrane potential. positively charged AA higher up compared to negatively charged AA lower down

What are some features that disrupt alpha helix

1. electrostatic repulsion

   • electrostatic interactions can stablize a helix.

   • Sequence of amino acids determine if it folds into helix

   • Low pH → lots of alpha helix since O=C-OH protonates

   • High pH → less alpha helix since O=C-O- deprotonates

   • Opposite charge = stablize, same charge = destabilize

2. Bulky R-groups such as isoleucine clash with each other

3. Small residues such as Gly and Ser favour conversion to the Beta conformation

   • Disruptive effect of amino acid on alpha helix stability

   • Proline is very high and very disruptive, glycine second, still decently disruptive, third alanine which is not disruptive

4. Proline disrupts the helix because the amide-N cannot become planar, it must retain sp3 shape.

   • also, the N has no H for H-bonding

   • Proline is often found at the ends of alpha helical segments because it imparts a sharp bend to the helix

   • Proline cannot be found in the centre of the helix. Proline very disruptive

Describe what a Beta-conformation is (B-pleated sheet)

1. Peptide bonds are planar

2. H-bonds foorm between residues in adjacent strands

3. R groups are above and below plane of sheet

4. amino acid preferences are highly context-dependent and generalizations are unhelpful and misleading

Whats a protein with high content of B-conformation

Silk or fibroin with an amino acid content if Gly, Ala, Ser and the remaining Amino acids.

how would you turn alpha keratin to beta conformation

steaming and stretching an alpha keratin (such as wool) (alpha helical) will convert it to Beta conformation but only temporarily.

Reminder, they are thermodynamically stable conformations and can be disrupted

an example would be hair perms where the straight hair is curled by locking the curl in place ny “Cys-S-S-Cys bonds (disulfide bonds)

• its like a ladder with s-s, the head and reduction separates the ladder into two strands, then recombined and curled in H2O2

Describe collagen (polyproline)

• It is a left-handed helix

• nothing to do with beta conformations

• Makes up 1/3 of the protein in an animal body, is a fibrous protein (provides strength) without alpha helix or beta sheet

• found in skin, bone, tendon, cartilage and teeth

• Collagen has its own secondary structure where it is an alpha chain which is built from three left-handed helixes, no interactions with H-bonds/no internal h-bonds but is stable

• in order for collagen to form, it requires both proline and hydroxyproline, makes collagen more durable as well

how is collagen stabilized

Chain is stabilzed by steric clashes of proline and hydroxy-proline side chains

• the three left handed chains are coiled are right hand twisted around each other and held together by interchain hydrogen bonds

• proline would rotate and clash, locking the structure in place

• the interior of the three chain coil is very packed - only glycine side chain fits

Describe the tertiary structure

• Secondary structure in a folded polypeptide and the rest of the chain are folded to give compact 3D structure

• About 40% of the AAs in a typical protein have hydrophobic (non-polar) R groups, like hydrocarbonds, interact unfavourably with water. Tertiary structure allows most of these R-groups to be buried in the centre of the protein, interacting with eachother (hydrophobic effect)

• most hydrophilic R groups are found on the surface, where they make favourable interactions with water'

  • With a hydrophilic exterior and hydrophobic interior, it is thermodynamically stable due to increase in water entropy

What is the main stabilizng feature of the tertiary structure?

Hydrophobic effect, where the hydrophobic amino acid side chains are buried in the interior of the protein.

Describe other stabilizng factors

1. Disulfide bonds (Cys-S-S-Cys) crosslink the protein with coovalent bonds

   • In reduced environemnt, in protein, less disulfide bonds

2. electrostatic or ionic interactions

   • R-groups with complementary charges form ionic bonds

   • Salt bridges are strongest in the interior of a protein - no water

3. H-bonds

   • Besides the regular patterns in secondary structures, can also occur between complementary R groups

   • H-bond in tertiary different than secondary as secondary, H-bonds are between functional group and side chains

4. Metal chelation

   • divalent cations like Mg2+, Ca2+, Zn2+ can play a structural role

Summary of tertiary structure features

• Proteins tend to be very compact with very few internal cavities or water molecules

• tertiary structures form so as to minimize the unfavourable interactions and maximize th efavourbale ones

Describe the Quaternary structure

• Describes the interaction of individual polypeptides already folded in their form

• The interaction of two or more polypeptides to form a multi-subunit protein

• tertiary = quaternary. need to ury hydrophobic groups, and ionic, H-bonding, disulfide formation, and metal chelation - shape is very important

What are polypeptides called when they are found in a multi-chain protein

subunits. a wide variety of quaternary structures are known, some large proteins having more than 10 different subunits

What is an example of the multisubunit protein

Hemoglobin. consisting of two alpha and two beta subunits whcih interact closely through complementary shapes, hydrophobic surfaces, ionic interactions, and H-bonds

Describe protein folding

• Folding is an ordered process that follow a series of steps tha are unique to each protein - the process is dependent on amino acid sequence

• Molecular chaperones (proteins that bind partly folded polypeptides) assist in folding. they stabilize key intermediates preventing non-specific aggregation and incorrect folding

• Only a limited number of folding motifs have been recognized

Describe protein denaturation

• Denaturation is the process by which a folded or native protein is converted to an unfolded form

1. The free energy difference between the native and unfolded states of a protein is usually small so mild treatments like heat or a change in solvent will denature most proteins

2. Once denatured, a few proteins can refold spontaneously, but this is rare (chaperones are usually required)

3. Most denatured proteins tend to aggregate and precipitate (ex. the proteins of egg white)

• harder to refold

What would happen if egg white was heated

the proteins denature: hydrophobic R groups are exposed to theh H2O and the proteins aggregate and become insoluble

What are the most common ways to denature proteins

• Heat is the most familiar denaturant (T>55C for most proteins) but pH (disrupts H-bonds and ionic bonds) and organic solvents also denature proteins

• High concentrations of certain solutes which disrupt the H-bonding system of water are excellent protein denaturants and keep the denatured form in solution

What are some proteins reffered to?

simple polypeptides, or are conjugated with suagrs (glycoproteins) or lipids (lipoporteins)

Others require nono-amino acid cofactors or prosthetic groups for full activity

• Cofactors may be inorganic (ex. metal ions or organic ( sugar, lipid, heme, flavin)

they may be covalently or non-covalently attached to the protein

enzyme cofactors are called coenzymes