Lecture 7 DA Flashcards
What determines the function of a protein?
Its structure.
What determines the conformation of a protein’s active site?
The conformation of the side chains of the protein’s non-active site regions.
Are non-active site region residues crucial to a protein’s function?
Yes, because they help correctly form the active site.
What are the aliphatic/polar amino acids?
Glycine Alanine Valine Leucine Methionine Isoleucine
Where are aliphatic/polar amino acids generally found, and why?
Typically inside the protein, usually not on the surface. Stabilises the protein’s structure by hydrophobic interactions, reducing free energy.
What are the aromatic amino acids? Which are hydrophobic/polar/non-polar?
Tyrosine - polar
Tryptophan - non-polar
Phenylalanine - very hydrophobic
Which amino acids strongly absorb UV light?
Aromatic
What are the polar/hydrophilic amino acids?
Serine Threonine Cysteine Proline Asparagine Glutamine
Where are polar/hydrophilic amino acids typically found?
On the surface.
Which amino acid is the only one able to form disulphide bridges?
Cysteine
What are the acidic amino acids? What charge do they have?
Aspartate
Glutamate
Both negative
What are the basic amino acids? What charge do they have?
Lysine
Asparagine
Histidine
All positive
Do peptide bonds have any degrees of freedom, or are they rigid? Do they contribute to 3D structure?
They are planar, with no degrees of freedom. Doesnt contribute to 3D structure, the phi and psi angles do.
Do all proteins form secondary structures?
Most, but not all.
What bonds are responsible for hydrogen bonds?
Hydrogen
Can the secondary structure of proteins be predicted by the primary structure?
Some regions, but not all.
What is the central paradigm?
Amino acid sequence of a protein determines its structure and function.
How can we tell if the primary sequence determines structure?
Protein can be denatured in urea, then allowed to renature. Some, but not all of the proteins will fold to its native state again.
What are three approaches to determining the structure-function relationship of a protein?
Predictive
Experimental
Protein peptide arrays - synthetic/expressed
What are two methods in the predictive approach to determining the structure-function relationship of a protein?
Sequence analysis
Modelling
What are two methods in the experimental approach to determining the structure-function relationship of a protein?
Site directed mutagenesis
Synthetic peptide analogues
What is looked for in a new protein sequence when determining function?
- Similar sequences (ie. BLAST)
- Distinctive patterns or domains with associated functions
- Secondary/tertiary structure predictions
- Motifs/residues important for function
- Physical properties - MW, pI, solubility etc
Describe the levels of protein structure/function relationship (6).
Superfamily Family Domain Motif Site Residue
Which levels of the protein structure/function relationship is associated with the secondary structure (3)?
Superfamily, family, and domain
What are some examples of protein structure from motifs or regions (3+)?
- Active sites
- Loops
- Interior of the protein
- Others include hydrophobicity, transmembrane regiosn, glycosylation sites, localisation signals, GPI anchors etc
What are the limitations of predicting protein structure/function by sequence analysis (2)?
- Doesn’t account for post-translational modifications such as glycosylation.
- pI calculations - assumes all side chains are accessible, but theyre not.
What are predictions of secondary structures based on (3)?
Statistical analysis
Physical properties
Amino acid distribution in known structures
How accurate are predictions of secondary structures?
Not very accurate, 55-65%
Newer methods are 70% accurate, still not enough.
How can predictions of secondary structures be improved?
Can incorporate information from multiple sequence alignments.
What is the best approach to predicting secondary structures?
Use a number of algorithms, and develop a consensus analysis.
Describe hydropathy analysis, and its use.
Sliding window analysis of a protein structure, to determine hydrophobic/philic regions.
Used to identify transmembrane sequences.
What do antigenicity algorithms predict, and what do they use to do so (4)?
Algorithms predict antibody epitopes, using hydrophilicity, side chain flexibility, surface probability, and predicted B-turns.
When determining domains and motifs, what can be detected (5+)?
- Post-translational modifications
- Domains
- Repeats
- DNA/RNA associated proteins
- Proteolytic cleavage sites
- Enzymes/transport/receptor proteins etc
What is a motif?
Small, highly conserved regions, shown as regular expressions.
Give an example of motif nomenclature.
[AG] x V x(2) {YW}
[] - means any amino acids in the bracket
x means any amino acid
V means only valine
x() means any amino acid by the number in the bracket
{} any amino acid except whats in the bracket
What do most proteins require for secretion? What happens to it? What is this entire process called?
Most have a signal peptide of 30-50 amino acids long. Cleaved off during secretion. Process called classical secretion.
Can classical secretion be predicted?
Yes.
Regarding chemical synthesis of proteins, what can be made (4)?
Proteins/peptides, which can be novel, toxic, or ones difficult to secrete, as well as hybrids.
What can chemically synthesised proteins be used for?
Epitope mapping.
Describe epitope mapping.
b
