New term: Active site labeling

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Kinetics of substrate analogue binding to an active site of an enzyme can be monitored to infer interaction with, eg, allosteric regulator. This is close to enzymatic assay but not exactly. It is also somewhat close to what footprinting is.

In general, any sort of labelling can be monitored to infer interaction with a partner that modifies labelling reaction. Both acitve site labeling and hydrogen exchange would fit such description. What about adding a general 'labelling assay' term (or maybe 'probe interaction assay' - see below) as a child of 'biochemical' and then adding 'active site labelling assay' as its child. Mass spectrometry study of hydrogen/deuterium exchange (MI:0944) could be another child of the 'labelling assay'. Some variants of MI:0417 (footprinting) would also fit here.

BTW: definition of footprinting:

'footprinting analysis is used to identify regions of molecules directly involved in binding other macromolecules and therefore protected from the effects of degradative enzymes.'

seems to be too detailed - there are already non-enzymatic children there; 'direct involvement' might be mostly true in case of DNA but is much harder to prove in case of proteins (interaction might exert its effects through allosteric effects).

For now all I need is 'active site labelling assay' to curate PMID:9751050 but I'd think in the long run it would make sense to group, into one CV branch, all terms describing methods that monitor reactions of 'probes' (hydrogen, small molecules, enzymes) in order to infer interactions between biomolecules... there are quite a few methods already in CV that seem to be very diverse but work according to the very same generic principle...

Reported by: lukasz99

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Currently we have the term "conditional site labelling', which has the related synonym "active site labelling". It is a child of proximity labelling technology, I believe we can possibly put the probe-specific terms uder it as well if the need comes. For now, I think you can use this to curate PMID:9751050, let me know if you feel it's not thecase.

And a proposal for a new definition of footprinting (I agree, the old definition was too detailed):

"Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

Let me know if that's fine for you and I'll update the CV accordingly.

Pablo.

Original comment by: pporras

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Currently we have the term "conditional site labelling', which has the related synonym "active site labelling". It is a child of proximity labelling technology, I believe we can possibly put the probe-specific terms uder it as well if the need comes. For now, I think you can use this to curate PMID:9751050, let me know if you feel it's not thecase.

Pablo, I'm afraid it won't cut it - 'proximity labelling technology' is defined as:

'Methods depend on a modification that only takes place with the close proximity of two molecules - a protein fused to the bait can then modify any neighbouring prey proteins, for example.'

and this is sort of opposite to what's in PMID:9751050 - I'm after fig 5b (cdk6/p16 case) where labeling of cdk6 active site is reduced in presence of p16

And a proposal for a new definition of footprinting (I agree, the old definition was too detailed):

"Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

Let me know if that's fine for you and I'll update the CV accordingly.

this one seems to be better :O) thanks, lukasz

Original comment by: pporras

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I'm afraid it won't cut it - 'proximity labelling technology' is defined as:

'Methods depend on a modification that only takes place with the close proximity of two molecules - a protein fused to the bait can then modify any neighbouring prey proteins, for example.'

and this is sort of opposite to what's in PMID:9751050 - I'm after fig 5b (cdk6/p16 case) where labeling of cdk6 active site is reduced in presence of p16

If I understood correctly, in figure 5b they are showing that presence of p16 somehow blocks the access of [14C]FSBA to the active site of Cdk6, right? You are probably right in pointing out that our definition actually goes in the direction of enabling labelling, rather than protecting from it, which seems to be the case. I would hesitate to use 'active site labelling' as a term name for this particular case, though, since it seems to have an implicitly "positive" meaning.

I propose creating a new term called 'site-labelling protection' (or something like that, I am terrible naming things), with a definition that would go as follows:

"The interaction is detected through selective, chemical blockage of a specific site -which can be an active site- that becomes inaccessible once the participants are interacting. The interaction is detected through loss of signal of the chemical label."

We can then create a parent term for 'labelling technology' (direct child of affinity technology) that would have this new term and the 'proximity labelling technology' as children.

Does that sound reasonable to you?

And a proposal for a new definition of footprinting (I agree, the old definition was too detailed):

"Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

Let me know if that's fine for you and I'll update the CV accordingly.

this one seems to be better :O) thanks, lukasz I'll update this along with the new terms once we reach a consensus.

Cheers,

Pablo.

Original comment by: pporras

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Hi Lukasz,

First, I put a copy of this and your previous email in sourceforge so everybody can follow the discussion.

Now to it.

I'm afraid it won't cut it - 'proximity labelling technology' is defined as:

'Methods depend on a modification that only takes place with the close proximity of two molecules - a protein fused to the bait can then modify any neighbouring prey proteins, for example.'

and this is sort of opposite to what's in PMID:9751050 - I'm after fig 5b (cdk6/p16 case) where labeling of cdk6 active site is reduced in presence of p16

If I understood correctly, in figure 5b they are showing that presence of p16 somehow blocks the access of [14C]FSBA to the active site of Cdk6, right? You are probably right in pointing out that our definition actually goes in the direction of enabling labelling, rather than protecting from it, which seems to be the case. I would hesitate to use 'active site labelling' as a term name for this particular case, though, since it seems to have an implicitly "positive" meaning.

I propose creating a new term called 'site-labelling protection' (or something like that, I am terrible naming things), with a definition that would go as follows:

" The interaction is detected through selective, chemical blockage of a specific site -which can be an active site- that becomes inaccessible once the participants are interacting. The interaction is detected through loss of signal of the chemical label."

We can then create a parent term for 'labelling technology' (direct child of affinity technology) that would have this new term and the 'proximity labelling technology' as children.

Does that sound reasonable to you?

And a proposal for a new definition of footprinting (I agree, the old definition was too detailed):

"Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

Let me know if that's fine for you and I'll update the CV accordingly.

this one seems to be better :O) thanks, lukasz I'll update this along with the new terms once we reach a consensus.

Cheers,

Pablo.

On 30/06/2015 16:30, Lukasz Salwinski wrote:

On 06/30/2015 07:37 AM, Pablo Porras wrote:

Currently we have the term "conditional site labelling', which has the related synonym "active site labelling". It is a child of proximity labelling technology, I believe we can possibly put the probe-specific terms uder it as well if the need comes. For now, I think you can use this to curate PMID:9751050, let me know if you feel it's not thecase.

Pablo, I'm afraid it won't cut it - 'proximity labelling technology' is defined as:

'Methods depend on a modification that only takes place with the close proximity of two molecules - a protein fused to the bait can then modify any neighbouring prey proteins, for example.'

and this is sort of opposite to what's in PMID:9751050 - I'm after fig 5b (cdk6/p16 case) where labeling of cdk6 active site is reduced in presence of p16

And a proposal for a new definition of footprinting (I agree, the old definition was too detailed):

"Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

Let me know if that's fine for you and I'll update the CV accordingly.

this one seems to be better :O) thanks, lukasz

Pablo.

---

\ [mi-controlled-vocab-changes:#381] New term: Active site labeling**

Status: open Group: Created: Wed Jun 17, 2015 03:07 AM UTC by Lukasz Salwinski Last Updated: Wed Jun 17, 2015 03:07 AM UTC Owner: nobody

Kinetics of substrate analogue binding to an active site of an enzyme can be monitored to infer interaction with, eg, allosteric regulator. This is close to enzymatic assay but not exactly. It is also somewhat close to what footprinting is.

In general, any sort of labelling can be monitored to infer interaction with a partner that modifies labelling reaction. Both acitve site labeling and hydrogen exchange would fit such description. What about adding a general 'labelling assay' term (or maybe 'probe interaction assay' - see below) as a child of 'biochemical' and then adding 'active site labelling assay' as its child. Mass spectrometry study of hydrogen/deuterium exchange (MI:0944) could be another child of the 'labelling assay'. Some variants of MI:0417 (footprinting) would also fit here.

BTW: definition of footprinting:

'footprinting analysis is used to identify regions of molecules directly involved in binding other macromolecules and therefore protected from the effects of degradative enzymes.'

seems to be too detailed - there are already non-enzymatic children there; 'direct involvement' might be mostly true in case of DNA but is much harder to prove in case of proteins (interaction might exert its effects through allosteric effects).

For now all I need is 'active site labelling assay' to curate PMID:9751050 but I'd think in the long run it would make sense to group, into one CV branch, all terms describing methods that monitor reactions of 'probes' (hydrogen, small molecules, enzymes) in order to infer interactions between biomolecules... there are quite a few methods already in CV that seem to be very diverse but work according to the very same generic principle...

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Pablo Porras Millán, Ph.D. IntAct Scientific Curator European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) Wellcome Trust Genome Campus Hinxton Cambridge CB10 1SD United Kingdom Tel: +44 1223 494482 email: pporras@ebi.ac.uk

URL: http://www.ebi.ac.uk/intact/main.xhtml

Original comment by: pporras

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Pablo, 'site-labelling protection' seems to be quite specific as it is applicable only to labels (conventionally thought of as molecules that specifically and covalently bind to proteins/nucleic acids) and only in cases where interaction slows the reaction (note that opposite effect wouldn't be too hard to demonstrate - eg for allosterically activated enzyme labelled at its active site). So what about a very generic 'probe interaction assay' term placed directly as a child of 'biochemical' (I'm somehow missing the logic behind 'affinity technology' branch so I'd rather avoid putting things there until someone clearly explains how to decide what goes into 'affinity technology' and what is to stay outside). it could be defined as (note, I'm also bad at writing definitions :o/:

'An assay that uses molecular probes, such as ions, small molecules or antibodies to monitor interactions between biomolecules under study'

'labelling assay' would be a child of 'probe interaction assay' defined as:

'The interaction is inferred from the effect it exerts on a selective chemical labelling of one of the interacting partners.'

this way we could cover labelling effects that go either way and also group some of the already existing terms (eg MI:0944:hydrogen/deuterium exchange, MI:0602:chemical footprinting and even MI:1087:monoclonal antibody blockade) that fit the generic description of 'probe interaction assay'

In principle 'labelling assay' can be further split into protection (to finally get to your 'site-labelling protection') & enhancement but, as the approach is rather infrequent, there's very little to gain this way...

Is there anyone willing to chip in a third party comment ? ;o)

lukasz

Original comment by: lukasz99

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'site-labelling protection' seems to be quite specific as it is applicable only to labels (conventionally thought of as molecules that specifically and covalently bind to proteins/nucleic acids) and only in cases where interaction slows the reaction (note that opposite effect wouldn't be too hard to demonstrate - eg for allosterically activated enzyme labelled at its active site).

Given that we already have a working term for the opposite effect ('conditional site labelling', MI:2168), I think it will still be consistent to create 'site-labelling protection', specific as it is. I think other strategies of exposing/protecting a site not involving small molecuels can be covered by existing specific terms or by your generic proposals commented below.

So what about a very generic 'probe interaction assay' term placed directly as a child of 'biochemical' (I'm somehow missing the logic behind 'affinity technology' branch so I'd rather avoid putting things there until someone clearly explains how to decide what goes into 'affinity technology' and what is to stay outside).

That's fine with me, I don't have any problem trying to make things a bit tidier and I agree that 'affinity technology' is a rather dodgy term.

it could be defined as (note, I'm also bad at writing definitions :o/:

'An assay that uses molecular probes, such as ions, small molecules or antibodies to monitor interactions between biomolecules under study'

Rather generic, but I guess it's fine.

'labelling assay' would be a child of 'probe interaction assay' defined as:

'The interaction is inferred from the effect it exerts on a selective chemical labelling of one of the interacting partners.'

Ok for me.

this way we could cover labelling effects that go either way and also group some of the already existing terms (eg MI:0944:hydrogen/deuterium exchange, MI:0602:chemical footprinting and even MI:1087:monoclonal antibody blockade) that fit the generic description of 'probe interaction assay'

Fine with me, I'll move them accordingly.

In principle 'labelling assay' can be further split into protection (to finally get to your 'site-labelling protection') & enhancement but, as the approach is rather infrequent, there's very little to gain this way...

As I mentioned above, we already have terms that would distinguish between enhancement/protection strategies. I'd leave them there, despite the potentially marginal gain.

Is there anyone willing to chip in a third party comment ? ;o)

Unless there are further comments, I'll do the changes as soon as we find an agreement in the coming days.

Cheers,

Pablo.

Original comment by: pporras

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ok, so it looks like we will end up with:

MI:0401:biochemical	probe interaction assay

labelling assay (with additional, double-parent siblings MI:0944:hydrogen/deuterium exchange, MI:0602:chemical footprinting, MI:1087:monoclonal antibody blockade) | site-labelling protection & MI:2168:conditional site labelling

or did I manage to mess it up ?

lukasz

Original comment by: lukasz99

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Hi. No, that's pretty much it. Just notice that 'conditional site labelling' currently goes under 'proximity labelling technology' (MI:1313), which I assume could perfectly go under labelling assay nevertheless, along with its 3 or 4 four cilhdren terms.

Unless there are any objections to this, I will then create the new terms and re-shuffle thing appropriatedly.

Cheers,

Pablo.

Original comment by: pporras

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Hi. No, that's pretty much it. Just notice that 'conditional site labelling' currently goes under 'proximity labelling technology' (MI:1313), which I assume could perfectly go under labelling assay nevertheless, along with its 3 or 4 four children terms.

oops... in this case I'd leave it out, at least for now. we can always add it later which is easier then removing the term - I'd rather read carefully through all the children terms before blindly adding them...

lukasz

Original comment by: lukasz99

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I have just commited the following 'site-labelling protection' to the CV. Please send feedback! Here's a summary:

1.- New terms 'probe interaction assay' (MI:2197) and 'labelling assay' (MI:2198) have been created, with the following definitions:

proximity interaction assay: An assay that uses molecular probes, such as ions, small molecules or antibodies to monitor interactions between biomolecules under study.

labelling assay (child of the previous one): The interaction is inferred from the effect it exerts on specific chemical labelling of one of the interacting partners.

2.- 'footprinting' definition updated to the following: "Footprinting analysis is used to identify regions of molecules involved in binding other macromolecules and therefore protected from the effects of degradative enzymes, chemical treatment or other deleterious treatments".

3.- I have created a new term for 'specific site-labelling protection', altering the name to distinguish it from 'chemical footprinting': "The interaction is detected through selective, chemical blockage of a specific site -which can be an active site- that becomes inaccessible once the participants are interacting. The interaction is detected through loss of signal of the chemical label".

4.- Notice that labelling assay and footprinting definitions have some degree of overlap. From y point of view, what differentiates methods under the 'labelling assay' branch is that they address one specific site at a time. Footprinting applies to technologies where the affected regions are not known a priori, so the treatment is not specific in the same way it is for labelling assays.

5.- Concerning moving MI:0944:hydrogen/deuterium exchange, MI:0602:chemical footprinting and MI:1087:monoclonal antibody blockade under the 'probe interaction assay' branch, I have only moved MI:1087 directly under 'probe interaction assay', since antibody treatment is supposed to be site/epitope-specific. The other two terms remain where they are, since I am not sure about the specificity of the assays in relation to the sites they affect.

6.- After careful reviewing of all children terms, 'proximity labelling technology' (MI:1313) and all its children are not any more under 'affinity technology', but under the new term 'labelling assay'.

7.- A mistake in the definition of 'chemical footprinting' has been amended. Where it said "Residues in close contact with the binding partner are protected from cleavage by the enzyme" it now reads "Residues in close contact with the binding partner are protected from chemical modification".

That's all for now, I am commiting the changes right now.

Cheers,

Pablo.

Original comment by: pporras

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• status: open --> closed
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Original comment by: pporras