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CCP4BB  October 2018

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Subject:

Re: Assumptions on protein activity (again)

From:

Tom Peat <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Fri, 12 Oct 2018 23:39:55 +0000

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Hello Markus, 

As has been mentioned by others, there are many ways to inactivate or inhibit a protein's activity. 
Why do you assume your protein is unfolded? From the information you have given, the protein is folded, just inactive or has low activity under several buffer conditions other than the one 'optimal' condition. 

Best regards, tom

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
[log in to unmask]

________________________________________
From: CCP4 bulletin board <[log in to unmask]> on behalf of Markus Heckmann <[log in to unmask]>
Sent: Friday, October 12, 2018 10:39 PM
To: [log in to unmask]
Subject: [ccp4bb] Assumptions on protein activity (again)

Dear all
Thanks to for all the responses. I would continue my question for
getting more advice.

I expressed a dimeric multi-protein (600 KDa).  This protein was
purified (affinity chromatography) in 4 different common buffers and
run with the same SEC-buffer. We observe clear single size-exclusion
peak referring to the dimer. We then measure activity of these 4
different protein yields in a well established assay.

What we observe: Only one out of 4 has high activity and even one
sample has NO activity at all. In all cases, the HPLC-SEC-MALS signal
using 'protein sample from activity assay' confirms unequivocally  a
dimeric state.

The observation of dimeric peak without activity means that  the
protein have a state that is either mis-folded - either locally or
partially. Are there any *sensitive* methods to detect this behaviour
- minor structural changes?

Can circular-dichroism detect these changes? or any other methods for
a large multi-domain protein?

Many thanks,
Markus



Previous responses:
-----------------------------------
I would not say *active* in the case of an enzyme, but probably
*folded*. An enzyme may have many conformational states, some of which
may represent inactive states, which will not be distinguished with
gel filtration (because their hydrodynamic radii will be roughly the
same), unless the inactivation involved unfolding and aggregation of
the protein.
--------------------------------
Is your enzyme pH sensitive? For example, if it has a histidine in the
active site and most of the buffer conditions you are testing are
below pH 6, you may be looking at a well folded protein that just
isn't active because you've protonated the active site residue. Or it
could be that the buffers you are testing are binding to your protein
and sterically interfering with your substrate? It doesn't mean that
your protein isn't folded or even inactive if you have just blocked
the binding site, merely inhibited. There could be all kinds of
reasons that changing buffers could change the activity of the protein
without unfolding the protein itself. Another example is that people
often use phosphate buffer in purification, but if the enzyme requires
a Mg, you could be inadvertently pulling that out of the enzyme by
using phosphate buffer (or using sulfate with an enzyme that requires
Ca, etc).
I'm sure it is possible that there are many enzymes in the PDB that
are clearly well folded (have good structures) that are not in their
fully active states due to the crystallisation conditions used to
obtain the crystals. We are usually capturing a single state of a
protein which usually has to be mobile to perform its enzymatic
function.
-----------------------
You can speak for yourself, but not for me. I do not assume activity
from a gel; that's what assays are for.Different buffers: it could be
you have a cofactor, perhaps a metal. The best practice is to document
what you do in your publications to the extent that a reader could
duplicate your results.
-----------------------
There are lots of examples in the PDB of incorrect structures. And a
single peak on SE doesnt guaruntee correctly folded protein. What were
the differences between the buffers? pH, ionic strength and additives
all matter for enzyme activity, and many buffers do bind to active
sites thus affecting activity (despite the general attempt to use
large molecules which are unlikely to bind in the cases of the Good
buffers). All that being said, the idea of a single, correctly folded
conformation of an enzyme/protein is an oversimplification used in
textbooks rather than the more complicated picture held by experts in
the field.
--------------------------
I believe the strong assumption in the community is that a clear
single peak of appropriate Mw is a clear indication of pure protein,
worthy intensive crystallization efforts. Whether it is active is
another question and this should be measured.For your analysis, it is
not important in which buffers the protein is not active, but whether
the protein you purified is active in the buffer (maybe without
precipitant) you used for crystallization.A single apo structure is
usually not enough to determine the catalytic mechanism of an enzyme,
you usually need some substrate-, transition state- product- (analog)
structures as well. If your protein is active in the crystallization
buffer and the ligand complexes make chemical sense, you can be pretty
sure that you have crystallized the right conformation.If your protein
is not active in the crystallization buffer, you must critically
analyze the structure, if it makes chemical sense and if you can
explain the absence of activity (e.g. pH far from optimum; inhibitor
bound in the active site). I am currently working on an enzyme who's
active site loves all kinds of substituted and unsubstituted
phosphates, sulfates etc. so it is not active in a wide range of
buffers like phosphate, MES, MOPS, HEPES etc. However, the crystal
structures still represent the active conformation, the active site is
just blocked by some buffer component.Other proteins (proteases) can
only be crystallized in an inactive form, since active they chew
themselves to pieces.

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