Leerdoelen Eiwittechnologie
Study task 1: Protein expression
Websites 3 (https://www.ebi.ac.uk/training/online/course/introduction-protein-classification-
ebi/protein-classification)
1. Explain the following terms and their relations: protein family, protein domain, proteins
feature, protein signature
Proteins that have diverged from a common ancestral gene are known as homologous (and therefore
usually have similar structures and functions).
Protein family: A group of proteins that share a common evolutionary origin, reflected by their
related functions and similarities in sequence or structure.
- Hierarchies = superfamily → subfamily
Protein domain: Distinct functional and/or structural units in a protein. Usually they are responsible
for a particular function or interaction, contributing to the overall role of a protein. Domains may
exist in a variety of biological contexts, where similar domains can be found in proteins with different
functions.
Sequence feature: Groups of amino acids that confer certain characteristics upon a protein and may
be important for its overall function.
- Active sites → catalytic activity
- Binding sites → binding molecules or ions
- Post-translational modification (PTM) sites → phosphorylated, palmitoylated, acetylated
- Repeat sites → structural properties
Protein signature: A set of tools used to classify proteins into families and to predict the presence of
important domains or sequence features.
- Multiple sequence alignment of proteins sharing a set of characteristics
- Signature types:
o Patterns
o Profiles
o Fingerprints
o Hidden Markov models (HMMs)
2. Name 4 different protein-protein interaction domains
- Src homology 2 (SH2) domain → recognizes phosphorylated tyrosines
o Plays a role in tyrosine receptor signaling
- Src homology 3 (SH3) domain → recognizes hydrophobic P – x – x – P sequence
- PTB domain → recognizes a NPxpY motif
o Binding specificity from residues amino-terminal to the (phosphor-) tyrosine
- PDZ domain → involved in specificity of receptor tyrosine kinase-mediated signalling
3. Interpret the molecular principle of a given protein-protein interaction domain
Chapter 6.4 – 6.7 (Analysis of genes and genomes)
4. Explain the two-hybrid screening technique to identify protein-protein interactions
The basis for two-hybrid interaction screening is the modular nature of eukaryotic RNA polymerase II
transcriptional activator proteins.
- A sequence-specific DNA binding domain (DBD) → to bind to promoters
- An activation domain (AD) → site of recruitment that results in gene expression
In many transcriptional activator proteins, the DBD and AD functions are found within the same
polypeptide but in separate and separable parts of the protein.
,If the DBD and AD can be linked in some way, protein-protein interaction of polypeptides fused to
them, then a functional activator protein in which the DBD is linked to the AD will be formed and
gene activation will occur.
5. Explain the roles of the functional domains of Gal4p in the two-hybrid screening
Gal4p is a single polypeptide comprising 881 amino acids and divided into distinct regions with the
DBD at the amino-terminal end and the AD at the carboxyl-terminal end of the protein.
If interaction between Gal4p DBD (X, bait) and Gal4p AD (Y, prey) → expression of reporter
6. Create functional fusion genes in the appropriate vectors for two-hybrid screening
, 7. Name 4 different reporter genes and their proteins
8. Explain the problems (and their solutions) with two-hybrid screening
- Protein folding
- Protein stability
- Weak interactions → Not strong enough interaction to recruit transcription
- False positives → transcription of the reporter without interaction between ‘’bait and prey’’
- Transcription factors
- In yeast cell nucleus
- Membrane proteins → Both ‘’bait and prey’’ must enter the nucleus, some will not be able to
do this
- Fortuitous interactions → Interactions that aren’t
physiologically relevant
- Bait protein cannot itself be a transcriptional
activator → The ‘’bait’’ itself is the activator
- Baits must be tested individually → A single ‘’bait’’
can screened using a library of interacting ‘’prey’’
partners, but the DBD-bait fusions must be
produced individually and transformed along with
the library into yeast cells.
Study task 1: Protein expression
Websites 3 (https://www.ebi.ac.uk/training/online/course/introduction-protein-classification-
ebi/protein-classification)
1. Explain the following terms and their relations: protein family, protein domain, proteins
feature, protein signature
Proteins that have diverged from a common ancestral gene are known as homologous (and therefore
usually have similar structures and functions).
Protein family: A group of proteins that share a common evolutionary origin, reflected by their
related functions and similarities in sequence or structure.
- Hierarchies = superfamily → subfamily
Protein domain: Distinct functional and/or structural units in a protein. Usually they are responsible
for a particular function or interaction, contributing to the overall role of a protein. Domains may
exist in a variety of biological contexts, where similar domains can be found in proteins with different
functions.
Sequence feature: Groups of amino acids that confer certain characteristics upon a protein and may
be important for its overall function.
- Active sites → catalytic activity
- Binding sites → binding molecules or ions
- Post-translational modification (PTM) sites → phosphorylated, palmitoylated, acetylated
- Repeat sites → structural properties
Protein signature: A set of tools used to classify proteins into families and to predict the presence of
important domains or sequence features.
- Multiple sequence alignment of proteins sharing a set of characteristics
- Signature types:
o Patterns
o Profiles
o Fingerprints
o Hidden Markov models (HMMs)
2. Name 4 different protein-protein interaction domains
- Src homology 2 (SH2) domain → recognizes phosphorylated tyrosines
o Plays a role in tyrosine receptor signaling
- Src homology 3 (SH3) domain → recognizes hydrophobic P – x – x – P sequence
- PTB domain → recognizes a NPxpY motif
o Binding specificity from residues amino-terminal to the (phosphor-) tyrosine
- PDZ domain → involved in specificity of receptor tyrosine kinase-mediated signalling
3. Interpret the molecular principle of a given protein-protein interaction domain
Chapter 6.4 – 6.7 (Analysis of genes and genomes)
4. Explain the two-hybrid screening technique to identify protein-protein interactions
The basis for two-hybrid interaction screening is the modular nature of eukaryotic RNA polymerase II
transcriptional activator proteins.
- A sequence-specific DNA binding domain (DBD) → to bind to promoters
- An activation domain (AD) → site of recruitment that results in gene expression
In many transcriptional activator proteins, the DBD and AD functions are found within the same
polypeptide but in separate and separable parts of the protein.
,If the DBD and AD can be linked in some way, protein-protein interaction of polypeptides fused to
them, then a functional activator protein in which the DBD is linked to the AD will be formed and
gene activation will occur.
5. Explain the roles of the functional domains of Gal4p in the two-hybrid screening
Gal4p is a single polypeptide comprising 881 amino acids and divided into distinct regions with the
DBD at the amino-terminal end and the AD at the carboxyl-terminal end of the protein.
If interaction between Gal4p DBD (X, bait) and Gal4p AD (Y, prey) → expression of reporter
6. Create functional fusion genes in the appropriate vectors for two-hybrid screening
, 7. Name 4 different reporter genes and their proteins
8. Explain the problems (and their solutions) with two-hybrid screening
- Protein folding
- Protein stability
- Weak interactions → Not strong enough interaction to recruit transcription
- False positives → transcription of the reporter without interaction between ‘’bait and prey’’
- Transcription factors
- In yeast cell nucleus
- Membrane proteins → Both ‘’bait and prey’’ must enter the nucleus, some will not be able to
do this
- Fortuitous interactions → Interactions that aren’t
physiologically relevant
- Bait protein cannot itself be a transcriptional
activator → The ‘’bait’’ itself is the activator
- Baits must be tested individually → A single ‘’bait’’
can screened using a library of interacting ‘’prey’’
partners, but the DBD-bait fusions must be
produced individually and transformed along with
the library into yeast cells.
