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<DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2>Chris<SPAN
class=200543601-23092002> (and all)</SPAN>,</FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2>You've caught me at a bad
time with this<SPAN class=200543601-23092002> query</SPAN>, as I am <SPAN
class=200543601-23092002>leaving tomorrow morning for a month-long trip to China
(and haven't finished packing yet)</SPAN>.</FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>It's unfortunately not very clear to me exactly how you are fitting the
data---are you always fitting as one species, are you always fitting the entire
range of sedimentation coefficients or just a window around the main peak, etc?
I would need to see the saved .fit files to really see what you have done. Your
documents also don't list the error bars on the numbers so it's hard to evaluate
whether the differences are even significant from that point of
view.</FONT></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2></FONT></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><SPAN
class=140341820-21092002><FONT size=2><SPAN class=140341820-21092002><SPAN
class=200543601-23092002><FONT size=2>One thing I would be concerned about is
that it seems likely you are including too many scans in the computation. Twenty
files is a <U>lot</U> for absorbance data, and indeed given the scan numbers
like 10-29 or 20-39 it sounds like you are including a large fraction of the
run, which sounds like it may be too many even for such a low molecular weight
protein. </FONT></SPAN></SPAN></FONT></SPAN></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><SPAN
class=140341820-21092002><FONT size=2><SPAN class=140341820-21092002><SPAN
class=200543601-23092002></SPAN></SPAN></FONT></SPAN></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><SPAN
class=140341820-21092002><FONT size=2><SPAN class=140341820-21092002><SPAN
class=200543601-23092002></SPAN></SPAN>Part of what you are asking appears to be
a technical question about the DCDT method and why the apparent sedimentation
coefficient varies depending on when you analyze the data during the run. This
is actually a (hopefully) well known phenomenon, especially for lower molecular
weight proteins. </FONT></SPAN></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><SPAN
class=140341820-21092002><FONT size=2></FONT></SPAN></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><SPAN
class=140341820-21092002>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>I will also point out that according to Walt Stafford's last statement on
the issue (see his RASMB e-mail at (<A
href="http://server1.bbri.org/rasmb_archives/1996/23.html"><FONT
size=2>http://server1.bbri.org/rasmb_archives/1996/23.html</FONT></A><FONT
size=2>) he says "<FONT color=#800000>Below about 15-17kDa it is nearly
impossible to get the boundary to clear the meniscus and so it is recommended
that the time derivative method not be used below that range</FONT>". I think
that is a bit extreme, but if you are working with a 13 kDa protein you will
definitely have systematic errors at some level---they are unavoidable, and
documented in my paper that is the basic reference for the program [<FONT
size=2>Philo, J. S. (2000). A method for directly fitting the time derivative of
sedimentation velocity data and an alternative algorithm for calculating
sedimentation coefficient distribution functions. <I>Anal. Biochem. </I>279,
151-163].</FONT></FONT></FONT></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2></FONT></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>The situation and basic source of the errors is slightly different
depending on whether one is fitting to the <EM>g(s*)</EM> data or to the
<EM>dc/dt</EM> data, with the <EM>dc/dt</EM> fits usually having somewhat
smaller errors on the sedimentation coefficient. Either way, even if you are
keeping the same total number of scans in the analysis, as you move that group
of scans earlier and later in the run you will be varying the degree
of peak broadening arising from the broad time span, and thus the
systematic error (see Fig. 6 in that paper).</FONT></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2></FONT></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002>
<DIV><SPAN class=140341820-21092002><FONT size=2>So<SPAN
class=200543601-23092002> </SPAN>the systematic shift in sedimentation
coefficient as you analyze scans from different times in the run is<SPAN
class=200543601-23092002>, at least in part,</SPAN> <U>an expected
result</U> based on the inherent approximations of this method. On top of that,
if (as is likely the case) you have more than one species in your sample, then
as you analyze the data later and later in the run you are 'losing' the faster
sedimenting components.</FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>You can of course do a numerical simulation of your experiment to
easily evaluate the degree to which this shift is expected (and I would
recommend doing that). Other sources of systematic shifts in sedimentation
coefficient as the run proceeds are errors in the meniscus position, or
temperature changes during the run.</FONT></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2>So, bottom line, yes, it is
possible to detect small changes in protein conformation with this approach, but
for proteins of this size you must be <U>very careful</U> and <U>must</U>
compare data at similar times in the run (and of course at a time when all the
relevant species are still entirely in the cell). <SPAN
class=200543601-23092002>Walter's difference sedimentation suggestion is of
course another good approach.</SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002></SPAN></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002>To give you an idea of the precision and
reproducibility that is possible, at least with larger proteins, and if one is
careful, here are some of my results from DCDT+ for the same protein
measured 4 times over 18 months:</SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002>
6.444 +/- 0.005 S</SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002>
6.448 +/- 0.005 S</SPAN></FONT></SPAN></SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002>
6.450 +/- 0.005
S</SPAN></FONT></SPAN></SPAN></FONT></SPAN></SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002>
6.450 +/- 0.005
S</SPAN></FONT></SPAN></SPAN></FONT></SPAN></SPAN></FONT></SPAN></SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2>Overall, though, I always say
that the most accurate sedimentation coefficients will never come from anyone's
DCDT implementation. The most accurate values come from whole boundary
approaches, like my SVEDBERG or <SPAN class=200543601-23092002>Peter
</SPAN>Schuck's SEDFIT or <SPAN class=200543601-23092002>Joachim
</SPAN>Behlke's LAMM<SPAN class=200543601-23092002> <EM>etc.</EM></SPAN>,
<U>because those approaches use data from the whole run, not just a small
portion</U><SPAN class=200543601-23092002>.</SPAN></FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2><SPAN
class=200543601-23092002></SPAN></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>With regard to the broader issue of whether this approach is a good one
for your experiments, I'm not sure of this but apparently you are always
analyzing the data as a single species. That may not always be
an appropriate model for these samples, and if it isn't then the results
are automatically at best suspect and at worst invalid. You seem to be assuming
that the protein is always a monomer, but isn't it likely that your aged sample
can form aggregates, not just change the conformation of the monomer? And don't
your refolded samples potentially contain two species, a folded and an unfolded
form (and maybe aggregates too, for that matter). </FONT>
<DIV><SPAN class=140341820-21092002></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2>You may want to consider
whether what you really want to report is an overall weight-average
sedimentation coefficient for the sample, rather than the value from a fit as a
particular species (which will not necessarily give you a thermodynamically
valid weight average if you have multiple conformations within that 'single
species').</FONT></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><FONT size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>We can perhaps discuss this further after my
return.</FONT></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2></FONT></SPAN></SPAN> </DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2>John</FONT></SPAN></SPAN></DIV></SPAN></SPAN></DIV></SPAN></SPAN></DIV>
<DIV><SPAN class=140341820-21092002><SPAN class=200543601-23092002><FONT
size=2></FONT></SPAN></SPAN> </DIV></SPAN></SPAN></SPAN></DIV></DIV>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV class=OutlookMessageHeader dir=ltr align=left><FONT face=Tahoma
size=2>-----Original Message-----<BR><B>From:</B>
rasmb-admin@rasmb-email.bbri.org
[mailto:rasmb-admin@rasmb-email.bbri.org]<B>On Behalf Of </B>Chin,
Christopher<BR><B>Sent:</B> Friday, September 20, 2002 11:55 AM<BR><B>To:</B>
John Phillo (E-mail)<BR><B>Cc:</B> Rasmb (E-mail)<BR><B>Subject:</B> [RASMB]
Can DCDT+ be used to detect small change in protein conformation? What is its
limit?<BR><BR></FONT></DIV>
<DIV><FONT size=2>
<DIV><FONT size=2>
<DIV><SPAN class=020583415-16092002><SPAN
class=990025517-16092002></SPAN>T<SPAN class=990025517-16092002>o
</SPAN>John<SPAN class=560111818-17092002>,</SPAN> <SPAN
class=990025517-16092002><SPAN class=560111818-17092002>all </SPAN>DCDT+
users<SPAN class=560111818-17092002> <SPAN class=820054219-19092002>and
</SPAN>all<SPAN class=990025517-16092002> </SPAN>interested colleagues<SPAN
class=560111818-17092002>, </SPAN></SPAN></SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002><SPAN class=990025517-16092002><SPAN
class=560111818-17092002><SPAN class=560111818-17092002></SPAN> </DIV>
<DIV><SPAN class=020583415-16092002><SPAN
class=560111818-17092002></SPAN></SPAN> </SPAN></SPAN></SPAN><SPAN
class=020583415-16092002>John, I would like to have your comment in the
following three experiments using RNAse<SPAN class=560111818-17092002>A
</SPAN>as a model and DCDT+ <SPAN class=990025517-16092002>as the method
</SPAN>to do data analysis.</SPAN></DIV>
<DIV><SPAN class=020583415-16092002></SPAN> </DIV>
<DIV><SPAN class=020583415-16092002>Experiment 1: <SPAN
class=560111818-17092002>The p</SPAN>urpose is to check the stability of
RNAse<SPAN class=560111818-17092002>A</SPAN><SPAN class=560111818-17092002>.
The comparison is made between t</SPAN>he protein <SPAN
class=560111818-17092002><SPAN class=820054219-19092002>left</SPAN> </SPAN>at
room temperature for <SPAN class=990025517-16092002>a
</SPAN>couple <SPAN class=820054219-19092002>of </SPAN>days
and <SPAN class=560111818-17092002>that </SPAN>stor<SPAN
class=820054219-19092002>age</SPAN> in the <SPAN
class=820054219-19092002>freezer.</SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002><SPAN class=020583415-16092002>Experiment
2: <SPAN class=990025517-16092002>Question: </SPAN><SPAN
class=990025517-16092002>Can <SPAN class=560111818-17092002>the
</SPAN>denatur<SPAN class=560111818-17092002>ed</SPAN></SPAN><SPAN
class=990025517-16092002> </SPAN>RNase<SPAN class=560111818-17092002>A<SPAN
class=820054219-19092002> </SPAN></SPAN>(has 4 s-s bond) in GuHCl <SPAN
class=990025517-16092002>be refolded back </SPAN>to its native state in the
absence <SPAN class=820054219-19092002>of </SPAN>reducing agent<SPAN
class=560111818-17092002>?</SPAN></SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002><SPAN class=020583415-16092002><SPAN
class=020583415-16092002>Experiment 3: <SPAN
class=990025517-16092002><SPAN class=990025517-16092002>Question: </SPAN><SPAN
class=990025517-16092002>Can <SPAN class=560111818-17092002>the
</SPAN></SPAN>denatur<SPAN class=560111818-17092002>ed</SPAN></SPAN><SPAN
class=990025517-16092002> </SPAN>RNase<SPAN class=560111818-17092002>A<SPAN
class=820054219-19092002> </SPAN></SPAN>(has 4 s-s bond) in GuHCl <SPAN
class=990025517-16092002>be refolded back </SPAN>to its native state in the
presence of reducing agent <SPAN
style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA">TBP
(Tri-n-butylphosphine)<SPAN
class=560111818-17092002>.</SPAN></SPAN></SPAN></SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002><SPAN class=020583415-16092002><SPAN
class=020583415-16092002><SPAN
style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><SPAN
class=560111818-17092002></SPAN></SPAN></SPAN></SPAN></SPAN> </DIV>
<DIV><SPAN class=990025517-16092002>The major issue here is how <SPAN
class=560111818-17092002>I </SPAN>can differentia<SPAN
class=560111818-17092002>te</SPAN> the<SPAN class=560111818-17092002> <B
style="mso-bidi-font-weight: normal"><I
style="mso-bidi-font-style: normal"><SPAN
style="FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol">D</SPAN></SPAN>S<o:p></o:p></I></B></SPAN> difference
due to scan cho<SPAN class=560111818-17092002>sen</SPAN> <SPAN
class=560111818-17092002>from</SPAN> <SPAN
class=560111818-17092002>the <B style="mso-bidi-font-weight: normal"><I
style="mso-bidi-font-style: normal"><SPAN
style="FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol">D</SPAN></SPAN>S<o:p></o:p></I></B><B
style="mso-bidi-font-weight: normal"><I
style="mso-bidi-font-style: normal"><SPAN
style="FONT-SIZE: 14pt; FONT-FAMILY: Symbol; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol; mso-bidi-font-size: 12.0pt; mso-bidi-font-family: 'Times New Roman'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol">
</SPAN></SPAN></I></B>difference due to </SPAN><SPAN
class=560111818-17092002>real</SPAN> experimental finding<SPAN
class=820054219-19092002> (between the control and the
sample)</SPAN>.</SPAN></DIV>
<DIV><SPAN class=990025517-16092002></SPAN> </DIV>
<DIV><SPAN class=020583415-16092002><SPAN
class=990025517-16092002></SPAN><FONT size=2>I<SPAN
class=990025517-16092002> am please<SPAN
class=560111818-17092002>d</SPAN> to say that</SPAN><SPAN
class=990025517-16092002>, using the criteria that I have stated<SPAN
class=820054219-19092002>,</SPAN><SPAN class=500034417-17092002><SPAN
class=560111818-17092002> e.g. m</SPAN>aximized or magnified the<SPAN
style="FONT-SIZE: 14pt; FONT-FAMILY: Symbol; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol; mso-bidi-font-size: 12.0pt; mso-bidi-font-family: 'Times New Roman'"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol"><STRONG><EM>
</EM></STRONG><FONT size=2><FONT face=Arial><B
style="mso-bidi-font-weight: normal"><I
style="mso-bidi-font-style: normal"><SPAN
style="FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol">D</SPAN></SPAN>S<SPAN
class=560111818-17092002> </SPAN></I></B></FONT><FONT face=Arial>difference
due to scan cho<SPAN
class=560111818-17092002>sen</SPAN> </FONT></FONT></SPAN></SPAN>(<SPAN
class=820054219-19092002>use</SPAN> a small<SPAN
class=560111818-17092002>er</SPAN> number for denominator<SPAN
class=560111818-17092002> when convert<SPAN
class=820054219-19092002>ed</SPAN></SPAN><SPAN class=560111818-17092002> to
%</SPAN>)<SPAN class=560111818-17092002>,</SPAN> <SPAN
style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><SPAN
class=560111818-17092002><FONT face=Arial
size=2>m</FONT></SPAN>inimized or play down the <FONT
face="Times New Roman"><FONT size=2><B style="mso-bidi-font-weight: normal"><I
style="mso-bidi-font-style: normal"><SPAN
style="FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN
style="mso-char-type: symbol; mso-symbol-font-family: Symbol">D</SPAN></SPAN>S<SPAN
class=560111818-17092002> </SPAN></I></B></FONT>difference<SPAN
class=560111818-17092002> between <SPAN class=820054219-19092002>the
</SPAN>control and the sample</SPAN> (<SPAN
class=820054219-19092002>use</SPAN> a large<SPAN
class=560111818-17092002>r</SPAN> number for denominator<SPAN
class=560111818-17092002> when convert<SPAN class=820054219-19092002>ed</SPAN>
to </SPAN><SPAN
class=560111818-17092002>%</SPAN> </FONT></SPAN></SPAN><FONT
face="Times New Roman" size=3><SPAN
class=500034417-17092002>)</SPAN></FONT></SPAN><SPAN
class=560111818-17092002><FONT face="Times New Roman" size=3>,</FONT></SPAN>
the results are <SPAN class=820054219-19092002>pretty
much </SPAN><SPAN class=820054219-19092002>I have</SPAN> expected.
Do you think I <SPAN class=990025517-16092002>try to push </SPAN>the
instrument limit to<SPAN class=990025517-16092002>o far to</SPAN> <SPAN
class=990025517-16092002>obtain</SPAN> these results by manipulat<SPAN
class=820054219-19092002>ing</SPAN> the <SPAN
class=990025517-16092002>treatment of data</SPAN>? </FONT></SPAN></DIV>
<DIV><SPAN class=020583415-16092002> </DIV>
<DIV><SPAN class=560111818-17092002>s20,w values reported here are the results
of fitting dcdt.</SPAN></DIV>
<DIV><SPAN class=560111818-17092002></SPAN> </DIV>
<DIV><SPAN class=020583415-16092002>Thank you in advance<SPAN
class=560111818-17092002> for your comment or advice.</SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002></SPAN> </DIV>
<DIV><SPAN class=020583415-16092002><SPAN
class=020583415-16092002>Chris</SPAN></SPAN></DIV>
<DIV><SPAN class=020583415-16092002></SPAN>
<P>--------------------------------------------------------------------------
<BR>Christopher Chin <BR>Manager, XLA-Analytical Ultracentrifugation facility
<BR>Sealy Center for Structural Biology <BR>HBC&G, 5.134 MRB.UTMB,
Galveston,T<SPAN class=560111818-17092002>X</SPAN> 77555-1055
<BR>cchin@utmb.edu, 409-772-1693, efax 708-585-1920
<BR>---------------------------------------------------------------------------
</P><BR></DIV></SPAN></FONT></DIV></FONT></DIV></BLOCKQUOTE></BODY></HTML>