Thank you. I was thinking along the lines of amination but was
concerned about the reactivity of the His nitrogen...
To add more information about the system: this is an untagged
thermophile protein, no His column used for purification. It was heated
to 65 degC during purification at pH 6.8 and crystallized at pH 8.0
imidazole buffer and calcium acetate present.
I believe the two mesomeric states shown by Dr. Skerra would require
that the His_N - C - amino_N - Calpha are positioned in a plane, but
this is not the case. The tetrahedral angle is approaching 90 degrees
instead. (I have included a small screenshot with refmac density after
refinement with a carbon atom as atom X. The temperature factor of this
carbon is similar to the surrounding atoms after refinement.)
I guess the geometry would argue for the bis-amine formation requiring
reduction, as suggested by Artem.
The bis-amine formation would have to go through the amidine
intermediate and maybe the N-term is flexible enough to accomodate the
planar group before reduction.
There is a somewhat similar reaction observed in the crosslinking of
collagen. There, a lysine is converted in an allysine (aldehyde instead
of terminal amino group). The allysine reacts with another lysine to
form an imine, which can be reduced with NADH to form a stable
crosslink. So, I guess if we assume that the His is reactive enough to
attack the formyl group and the N-term is flexible enough to accomodate
the amidine then a crosslink by this mechanism is at least conceivable.
I will submit a sample to mass-spec analysis to verify the presence of
an additional carbon atom.
Thanks for the advice.
Gerwald
Prof. Dr. Arne Skerra wrote:
Dear Colleagues,
The amidine originating from the condensation reaction between the Met
N-formyl group and the His side chain should be the other way around!
This would also be roughly consistent with the reported geometry. In
fact, there should be two mesomeric states, provided there is N
protonation.
Arne Skerra
At 5:56 Uhr -0400 10.10.2008, Artem Evdokimov wrote:
If you're 100% sure that this is only one
atom then amination comes to mind.
I have no clue what conditions would favor such reactivity but it is
possible that the formyl group on the Met was aminated with the cyclic
N of
the histidine, resulting in either a substituted bis-amine (requires
reduction, may not be stable) or in a cyclic amidine (the latter
requires
four atoms - N1-C=N-Ca - to be in the same plane). This amidine should
have
some pretty interesting properties (probably more like a Schiff base
than an
amidine).
Could you tell us a bit more about the system you're working with?
Artem
P.S. The bond lengths you describe are not typical for C-N, however at
1.9A
resolution it is not very likely that the values you measured actually
correspond to the bond lengths (hopefully, because otherwise you have
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
gerwald jogl
Sent: Thursday, October 09, 2008 4:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Potential N-terminal cyclization
Hi All,
I am looking for some input regarding an unusual feature in one of our
structures. Maybe someone has come across something similar or has some
thoughts about it...
We have a 1.9 A crystal structure with well-defined density in the
N-terminal region. The side chain of His3 is oriented towards the
N-terminal amino group of Met1 and there is a nice difference density
'ball' right in between the His and the amino group suggesting that
there is an additional atom that is covalently linked. There are two
molecules in the asu and both show the same feature (no ncs
refinement).
My problem is that I cannot come up with a reaction that would result
in
such a linkage and there is not much to be found in the literature.
If I place a hypothetical atom in the difference density peak, I can
measure distances and angles. Here are some numbers: Distance from atom
X to the N-terminal nitrogen 1.72 (1.64 in chain B). Distance from atom
X to His NE2 1.58 (1.45 in chain B; the temperature factors of the His
side chain are slightly more consistent with this ring orientation
compared to the 180 degree flip that would bring CE1 towards atom X).
Atom X is coplanar with the His ring. The angle between NE2 - X - N1 is
95 deg (106 in chain B). The angle between X - N1 - Calpha1 is 111
degrees in both chains. As the N-terminal methionine is still present,
it is possible that the methionine formyl-group was present before the
hypothetical reaction. However, there is only one 'atom' in the
difference density.
Any comments or suggestions would be highly welcome.
Gerwald
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