Topic 3

What are peptide bonds?

• Peptide bonds are how amino acids are connected to make proteins 

How are peptide bonds formed? 

• The bond is formed between the alpha amino group on one amino acid and the alpha carboxyl group of another acid by a condensation rxn

What is unique about the polarity of the amino acid? 

• The two ends of the amino acid have different polarity 

• One end has a free amino group called N terminus, the other has a free carboxyl group called C terminus 

What is unique about the peptide bond single bond and what does it result in? 

• Peptide bond is written as a single bond, but actually has some characteristics of a double bond because of the resonance between the C-O and C-N bonds

• This constrains the flexibility of the chain and prevents some folding patterns

What are the four levels of protein structure? 

1. Primary structure: sequence of amino acids 

2. Secondary structure: spatial arrangement of a polypep. backbone 

3. Tertiary structure: 3D structure of entire polypeptide including all side chains 

4. Quaternary structure: spatial arrangement of polypeptide chains in a protein w/ multiple subunits 

What is the only bond that forms the backbone of a protein?

• Covalent bond

What is the secondary structure made up of and what are the two major types?

• Hydrogen bonds between N-H and C=O 

• 2 major types: alpha helix and beta pleated sheets

Explain the structure of the alpha helix bond

• Hydrogen bonds stabilize structure 

• Rigid cylinder structure w/ helical arrangement 

Explain the structure of beta pleated sheets

• Polypeptide chain folds in on itself creating parallel or an antiparallel βsheet, or mixtures of the two 

What 3 things determines whether a particular part of a sequence will fold into one or the other of these structures?

• interactions between side chains of the residues in the polypeptide

1. Steric hindrance between nearby side chains 

2. Charge repulsion between similarly charged side chains 

3. Presence of glycine and proline 

How does the presence of glycine and proline affect the sequence fold? 

Proline: Contains a ring that constrains bond angles, so it does not fit a-helix or b-sheet

Glycine: Due to the side chains flexibility, it interrupts secondary structures 

How does a tertiary structure form and what forces take part? Which is the most important one?

• Results from interactions between side chains 

• Stabilized by ionic bonds between side chains, hydrogen bonds, and van der Waals forces

• The most important force in the tertiary structure is hydrophobic effect

• Contains disulfide bonds

What is the hydrophobic effect?

• major driving force behind protein folding

• Hydrophobic (nonpolar) amino acid side chains avoid water and become buried in the interior of the protein

• while hydrophilic (polar) side chains remain exposed to the surrounding water. 

• This allows the protein to fold into a more stable, compact structure.

What are chaperone proteins?

• Proteins that prevent misfolding and aggregation of polypeptide chains 

What does this image show? 

• Partially folded protein enters chamber 

• Chamber is enclosed and protein is isolated 

• This allows protein to have time to fold correctly 

• Then released once correct

How does a quaternary structure form and what forces take part?

• Same ones as tertiary 

How does a protein naturally form its structure?

• All of its protein folding information is present within primary structure of the protein 

• If soaked with urea so that the protein unfolds, it will refold if the urea was removed

Protein domain vs Protein family

• Protein domains are independently folding, provides function and structure within a single protein chain

• Protein families are a group of entire proteins that share a common evolutionary ancestor and similar functions or sequences 

What are the different column chromatography approaches?

1. Gel filtration ( separation by size) 

2. Ion exchange (separation by charge) 

3. Affinity chromatography (separation by molecular interactions) 

How does Gel filtration work?

Mechanism: The column is packed with porous gel beads.

• Large molecules cannot fit inside the pores (excluded) →take the short path around the beads → are extracted first.

• Small molecules enter the internal maze of the beads → get delayed → are extracted last.

Key Factor: Elution speed depends on molecular size and protein shape (hydrodynamic radius).

How does Ion Exchange work? 

Ion Exchange Chromatography separates proteins by charge.

• Binding: Opposite charges attract—negatively charged proteins stick to a positively charged resin (like DEAE). 

• Therefore, uncharged proteins and cations will flow through because they do not stick 

• Elution: Increasing the salt concentration provides competing ions (e.g., Cl-) that knock the bound proteins off the resin so they flow out.

What does this picture explain

• Explains how a protein's net charge is controlled by the buffer's pH relative to the protein's pI which determines how it behaves in Ion Exchange Chromatography. 

What are the three scenarios of pI? 

1. pH = pI ; The net charge on the protein is 0. The protein will not stick to either anion or cation exchange resin 

2. pH < pI ; Net charge is positive due to more acidic environment (use cation exchange)

3. pH > pI ; Net change is negative due to more basic environment (use anion exchange) 

How does Affinity chromatography work? 

• Proteins are separated by using a highly specific interaction between a target protein and its corresponding ligand 

Steps: 

Binding: A specific ligand anchored to column beads traps only the target protein.

Washing: All non-target proteins flow straight through and are washed away.

Requirement: Requires prior knowledge of a ligand that specifically binds the protein of interest

What is SDS, and what roles does it play in SDS-PAGE?

SDS = an ionic detergent

• used to denature proteins

• used to bind to proteins to give them negative charge

How are proteins separated during SDS-PAGE, and which direction do they move?

• Separates purely by molecular mass / polypeptide chain size.

• Charged molecules move towards positive electrode

• Smaller molecules move faster toward the bottom of the gel

What is mass spectrometry used for?

• To characterize proteins in complex mixtures

• Can provide info on identity and abundance

How do we determine 3D structures of proteins using X ray Crystallography?

1. Purify protein

2. Crystalize protein

3. When X ray is applied, electrons scatter producing diffraction pattern

4. the diffraction pattern is used to build 3D protein structure model

What is NMR spectroscopy

Another method of building 3D protein

• Crystallization of protein is not required

When would we use NMR spec. vs X ray Crystallography?

NMR spec: for small proteins

X ray: For big proteins