JW - Protein Architecture II Flashcards
What is the typical structure of most globular proteins?
Most globular proteins have a domain structure
How many domains are present in the cell surface protein CD4?
4 IG domains
What is an example of a conserved domain?
EGF
- Conserved in many different protein structures (Neu, EGF precursor and TPA)
How are domains identified?
Using amino acid sequence profiles
Domain vs Module vs Repeat
Domain: a folded structural unit; the sequence need not be contiguous
- e.g. MHC II
Module: a domain with a contiguous sequence, repeatedly used in diverse proteins
- e.g. F3
Repeat: a unit that does not fold in isolation; several copies are needed
- e.g. Leu-rich repeat
What are some examples of popular protein domains? (7)
- IG
- F3
- F1
- EGF
- SH2
- SH3
- Kinase domain
What are antibodies made from?
IG domains
Describe the structure of IG domains (2)
IG domain:
- Around 100 residues
- 2 layered sandwich of 7( to 9) anti parallel b-strands, in two b-sheets with Greek key motif
What are the 2 main variants present in IG domains?
C1 variant:
- 4 stranded b-sheet DEBA & 3 stranded b-sheet GFC
C2 variant:
- 3 stranded b-sheet EBA & 4 stranded b-sheet GFCC’
- C’ strand switched sheet with respect to C1 variant
What is the function and structure of the MHC class I protein?
Function:
- presents antigens (peptides 8-10 residues) to T lymphocytes
Structure:
- non-covalent heterodimer
- a-chain: contains a3-domain and peptide binding domain
- b-chain: b2m (single IG-domain)
Two IG domains: a3 and b2m
What is Titin?
Giant muscle protein
- 90% IG or IG-like domains
What do interdomain linkers act as?
Hinges
- Passive elements that flexibly connect the functional domains
What do unstructured protein regions contain?
Binding motifs
- short and unstructured
- multiple specific protein binding motifs
What are Integrins?
Family of cell adhesion receptors, bind to extracellular matrix components
What are
Intrinsically Disordered Proteins (IDP) and what are their features? (2)
Parts of protein sequences or entire proteins that lack a defined 3D structure
- They may be entirely random coil or partially structured
- They carry out their function as unstructured proteins
What are 2 prominent examples of IDPs?
a-synuclein: human 140aa, functions in synaptic transmission
different structural features depending on environment:
- lipid bound
- Tetramer
- beta rich
- fibrilar state
Tau(s): stabilizes microtubules, phosphorylation state disease related
What is an example of a protein that contains both folded domains and unstructured regions?
P53: tumor suppressor protein contains both folded domains and IDPs
IDPs:
- TAD1 transactivation region
- PRR proline rich
- TD tetramerisaion region
- RD regulatory region
How do we study primary, secondary and tertiary structures of proteins?
1) Primary Structure
Chemically; Edman degradation
- Sequence of amino acids
Mass spectrometry
- Identification of protein fragments
2) Secondary and tertiary structures
- There is an array of methods available
What is a formula involed in a spectroscopic method for determining protein structure?
Beer-Lambert Law
A = e l c
e: molar extinction coefficient
l: path length in cuvette
c: concentration of protein (DNA,…)
What is Circular Dichromism (CD)?
CD is the unequal absorption of left-handed and right-handed circularly polarized light
- In proteins, the peptide bond is an optically active group giving rise to circular dichroism which in turn is sensitive to protein conformation
Helps determine secondary structure of a protein
Features of Electron Microscopy ? (5)
- Images of single particles is possible (limit resolution ~20A)
- Averaging or crystalline samples resolution ~4A) needs extensive image processing
- Routine use of EM on “ metal stained” samples for increased contrast
- Without metal stain but sample freezing leeds to fewer artefacts
- Whole cells can be imaged and distribution of proteins analysed inside the cell
What are 2 major limitations of Electron Microscopy?
- Radiation damage
- In transmission EM very thin samples needed
(~<500nm)
