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MIMS > Proteins and Molecular Recognition > Flashcards

Proteins and Molecular Recognition Flashcards

1
Q

type of contacts usually involved in permanent interactions between binding partners?

A

hydrophobic contacts so separation is energetically unfavourable in aqueous environment

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2
Q

type of surfaces usually involved in transient interactions between binding partners?

A

hydrophilic surfaces as the binding partners need to remain soluble when not in the complex

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3
Q

common features of protein-other molecule interactions?

A

highly specific, optimal affinity, tightly controlled

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4
Q

how can affinity of an interaction be quantified?

A

by the dissociation constant which is the ratio of the concentrations of the components of the complex/the concentration of the complex in the solution

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5
Q

what is the fraction of protein bound to a ligand in a solution equal to?

A

concentration of bound protein (conc. of the complex)/total protein concentration or if ligand concentration much higher than protein concentration, the total ligand concentration/total ligand concentration + the dissociation constant

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6
Q

what is a ‘Biacore’ instrument used for?

A

measurement of molecules associating with a sensor surface- surface plasmon resonance

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7
Q

what is surface plasmon resonance?

A

method for measuring affinity of interactions. one partner immobilised on sensor surface, solution of other partner passed over it. binding causes changes in refractive index at surface which is measured by the instrument in real time- can then calculate the on rate from this. then wash phase when dissociation of complex measure, off rate constant can be calculated

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8
Q

how does growth factor signaling work?

A

growth factor binds to a GF receptor, causes receptor dimerisation. this allows the intracellular domains of the receptor to phosphorylate each other creating scaffold for binding of proteins like Grb2 and Sos- recruited to membrane surface bringing Sos into contact with Ras (G protein). this activates Ras, which stimulates kinase cascade, one of final outcomes = activation of Fos and Jun, bind DNA sequences promoting transcription

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9
Q

what are Fos and Jun?

A

transcription factors which bind to specific DNA sequences and promote gene expression

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10
Q

are mutations that lead to interactions in GF signaling cascade in absence of GF oncogenic or tumour suppressor mutations?

A

oncogenic

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11
Q

what are SH3 domains?

A

small modular domains that drive protein:protein interactions. 50-75 amino acids long, have a groove on their surface which binds specific proline-rich sequences on partner proteins

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12
Q

describe the interaction between Grb2 (growth receptor binding protein) and Sos (nucleotide exchange factor)?

A

Grb2 has 2 SH3 domains that act as protein recognition modules, one of them interacts specifically with a region of Sos, binds to 9 residue proline rich peptide from Sos. peptide binding surface of Grb2 forms shallow binding pocket with complementary shape + surface properties to Sos peptide. small binding interface but high hydrophobic component so strong interaction. also Sos peptide forms H bonds, van der Waals and electrostatic interaction with SH3 domain

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13
Q

how is binding of proteins to DNA commonly achieved?

A

by positioning α-helices into the major groove of DNA where amino acid side chains on binding face of the α-helix can make specific H bonds with the DNA bases so the protein can recognise a particular DNA base sequence using the amino acid sequence of the recognition helix

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14
Q

what is found in both Fos and Jun?

A

α-helical DNA binding regions that form heterodimer that binds to specific DNA sequence

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15
Q

what are the methods of control of molecular recognition?

A

control through conformational change, control through phosphorylation

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16
Q

structure of the Ras-Sos complex?

A

small hydrophobic core of 3 side chains buried, rest of the large interface is hydrophilic with multiple H bonds and electrostatic interactions

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17
Q

effect of binding of Ras to Sos on Ras?

A

large conformational change- GTP binding pocket of Ras opens up releasing GDP so GTP can bind

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18
Q

how is phosphorylation used to control molecular recognition?

A

addition of phosphate to surfaces involved in recognition in TK linked pathways generates surface that is complementary to a binding partner resulting in signaling event

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19
Q

example of molecule that uses phosphorylation for molecular recognition?

A

SH2 domains require phosphorylation of Tyr residues on their binding partners

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20
Q

how many amino acids do SH2 domains contain?

A

around 100

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21
Q

example of an SH2 domain?

A

SH2 domain in Grb2 has 2 binding pockets, binds to specific phosphorylated peptide from the tyrosine kinase linked EGF receptor which interacts with both of the binding pockets- 1 pocket only makes tight interactions with phosphorylated Tyr residue- determines affinity, 2nd pocket binds to Asp residue- this determines specificity

22
Q

what does co-immunoprecipitation do?

A

uses antibodies to capture a protein of interest and any associated partner proteins from cell or tissue lysates to identify interacting partners. requires availability of an antibody which can recognise protein of interest

23
Q

how can the identity of the associated proteins captured in co-immunoprecipitation be identified?

A

using Western blotting or mass spectrometry

24
Q

what is the interactome?

A

the complete set of molecular interactions in a system

25
Q

function of surface plasmon resonance?

A

quantitates binding interactions to see likelihood of interactions occurring

26
Q

constant domains in antibodies?

A

2 in the arms and 4 in the stalk (Fc region)

27
Q

functions of the constant domains in antibodies?

A

interact with receptors on surface of cells leading to phagocytosis, interact with soluble complement proteins leading to cytotoxicity

28
Q

what are the 5 classes of immunoglobulin?

A

IgG, IgM, IgA, IgD, IgE

29
Q

most common immunoglobulin class?

A

IgG

30
Q

how many Y shaped units does immunoglobulin consist of?

A

1

31
Q

2nd most common immunoglobulin class, how many Y shaped units, what is its main role?

A

IgM, 5 units, first Ig made in response to an antigen, particularly effective against microorganisms

32
Q

major Ig in secretions like tears, saliva and milk, how many Y shaped subunits generally?

A

IgA, generally 2

33
Q

2 least abundant immunoglobulins, how many subunits, which is important in allergies + parasitic worm response?

A

IgD and IgE, 1 subunit, IgE is important for this

34
Q

what is influenza haemagglutinin? how do antibodies target it?

A

viral surface molecule that is needed for the virus to invade host cells as it recognises the host cells and promotes membrane fusion. antibodies bind to haemagglutinin and cover the host cell binding site

35
Q

uses of antibodies in research?

A

the ELISA, co-immunoprecipitation, western blots, immunohistochemistry

36
Q

how do Western blots work?

A

run SDS-PAGE, then transfer the proteins from the gel onto the surface of a sheet of nitrocellulose or a polymer. sheet can be added to solution containing antibodies to a particular protein, if protein present a band will appear when the antibody binding is detected at the position of the protein on the gel, can estimate size of protein by comparison to molecular weight standards

37
Q

what is a monoclonal antibody?

A

a single antibody type with a single structure that recognises a single epitope

38
Q

what is hybridoma technology?

A

B lymphocytes taken from immunised animal- some of them produce desired antibody but alone can’t grow and divide in culture. cells from mouse myeloma (B cell tumour) divide in culture indefinitely but don’t produce required antibody. B lymphocytes fused with myeloma cells, resultant hybridomas can grow and divide in culture and secrete antibodies which can be screened to find ones that produce desired monoclonal antibody

39
Q

why is hybridoma technology impact limited in humans?

A

cause human anti-mouse antibody response and reduced interaction with human receptors reduce their half-life and limit their efficacy

40
Q

how are mouse antibodies made more human-like?

A

take CDR loops from mouse antibody and graft them onto human variable domain framework to generate antibody with binding properties of mouse antibody but that is recognised as human by human immune system

41
Q

how can antibodies be generated directly from human variable domain genes using transgenic mice?

A

replace mouse VH and VL gene repertoires with human VH and VL genes so upon immunisation the mice generate antibodies with human variable domains

42
Q

how are human antibodies generated using antibody phage display?

A

genes encoding antibody VH and VL domains are cloned into the genome of a filamentous bacteriophage of E. coli, gives rise to individual phage particles that display a single functional antibody on the outside while carrying the gene encoding the antibody on the inside- can select antibody VH and VL genes with desired binding properties, once isolated antibody gene can be shuttled into variety of expression formats

43
Q

what is Humira (adalimumab)?

A

a very successful anti-TNF human antibody derived from phage display, used to treat rheumatoid arthritis

44
Q

what is Trastuzumab (Herceptin)?

A

a monoclonal antibody that binds to and blocks signaling of HER2 (human EGFR 2) which is over-expressed in 30% of breast cancer cases. Herceptin therefor blocks cell division and marks cancerous cells for immune destruction

45
Q

what is Cetuximab?

A

a chimaeric antibody drug used to treat colorectal cancer + head and neck cancers

46
Q

what is Pertuzumab?

A

humanised murine antibody used to treat breast cancer

47
Q

what is Panitumumab?

A

humanised murine antibody used to treat colorectal cancer

48
Q

what is Tarceva?

A

molecule that competitively inhibits the EGF receptor tyrosine kinase- competes with ATP, has higher binding affinity than ATP.

49
Q

what is Raf?

A

a Ser/Thr kinase activated by Ras-GTP by phosphorylation allowing ATP to bind

50
Q

mutation in Raf in melanoma?

A

a Val in the Raf activation loop is changed into Glu, which has negative charge mimicking phosphorylation so the Raf kinase is active in absence of the Ras that activates it

MIMS (15 decks)

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