Hi James,
We used to talk about primary and secondary radiation damage. The former
operates at room temperature where free radicals were said to be formed
in solution and diffuse around to damage proteins. Under cryoconditions
this no longer happens, leading to greatly improved crystal life time,
but we still have primary radiation damage, with the photons directly
hitting the protein.
It was my understanding that this was still considered to form a free
radical at the affected atom without there being any diffusion involved.
Sulfur atoms would be more sensitive as they have a larger X-ray
cross-section or because they may act as free-radical sinks where free
radicals generated nearby strip an electron from the sulfur, thereby
satisfying their own electronic configuration and converting the sulfur
into a radical state. For instance, in ribonucleotide reductase a
tyrosine free radical is formed spontaneously (using oxygen and an
dinuclear iron site) and "jumps" over 20 Angstrom from one subunit to
another to form a thiyl free radical in the active site. It then "jumps"
back to the tyrosine upon completion of the catalytic cycle. Although we
don't know how it jumps, certainly not by diffusion, there is general
agreement that it does happen.
People have also observed broken disulfides in cryocrystal structures
with the sulfurs at a distance that is too long for a disulfide but too
short for a normal non-bonded sulfur-sulfur interaction. I seem to
remember that this distance was suggested to indicate the presence of a
thiyl free radical. I'm no chemist of physicist so can't evaluate if
that claim is reasonable but if it is then that would be direct evidence
to support the involvement of a free radical state in radiation damage.
So I guess my questions/comments are
- what are the great many good reasons to think that free radicals do
NOT play a role in radiation damage under cryo.
- although diffusion does not happen below 130K, radicals do appear to
teleport, at least over short distances.
Bart
James Holton wrote:
I don't mean to single anyone out, but the assignment of "free radicals"
as the species mediating radiation damage at cryo temperatures is a "pet
peeve" of mine. Free radicals have been shown to mediate damage at room
temperature (and there is a VERY large body of literature on this), but
there are a great many good reasons to think that free radicals do NOT
play a role in radiation damage under cryo.
This "assignment" of free radicals to damage is often made (flippantly)
in the literature, but I feel a strong need to point out that there is
NO EVIDENCE of a free radical diffusion mechanism for radiation damage
below ~130K. To the contrary there is a great deal of evidence that
water, buffers and protein crystals below ~130 K are in a state of
matter known as a "solid", and molecules (such as free radicals) do not
diffuse through solids (except on geological timescales). If you are
worried that the x-ray beam is heating your crystal to >130 K, then have
a look at Snell et. al. JSR 14 109-15 (2007). They showed quite
convincingly that this just can't happen for anything but the most
exotic situations.
There is evidence, however, of energy transfer taking place between
different regions of the crystal, but energy transfer does not require
molecular diffusion or any other kind of mass transport. In fact,
solid-state chemistry is generally mediated by cascading
neighbor-to-neighbor reactions that do not involve "diffusion" in the
traditional sense. Electricity is an example of this kind of chemistry,
and these reactions are a LOT faster than diffusion. The closest
analogy to "diffusion" is that the propagating reaction can be seen as a
"species" of sorts that is moving around inside the sample. Entities
like this are formally called quasiparticles. Some quasiparticles are
charged, but others are not. If you don't know what a quasiparticle is,
you can look them up in wikipedia.
Some have tried to rescue the "free radical" statements about radiation
damage by claiming that individual electrons are "radicals". I guess
this must come from the "pressure" of such a large body of free-radical
literature at room temperature. However, IMHO this is about as useful
as declaring that every chemical reaction is a "free radical" reaction
(since they involve the movement of electrons). I think it best that
we try to call the chemistry what it is and try to stamp out rumors that
mechanisms are known when in reality they are not.
Just my little rant.
-James Holton
MAD Scientist
--
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Bart Hazes (Assistant Professor)
Dept. of Medical Microbiology & Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone: 1-780-492-0042
fax: 1-780-492-7521
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