From: Michael Bovee <mbovee@zoo.uvm.edu>
  To  : RASMB@BBRI.HARVARD.EDU
  Date: Tue, 9 Mar 1999 11:05:48 -0500

Using Nonlin (Mac or PC)

Hello RASMBers,

I'm looking for a few pointers on using Nonlin to estimate molecular mass
for a hetero-associated complex:

The question I want to address is whether our histidyl-tRNA synthetase
(from E. coli,  a stable homodimer in solution)  binds a tRNA in only one
or both active sites simultaneously.

Here's what I've learned from sed velocity:
The free enzyme (homodimer) has a s* of 5.5 at pH 7.4,  6.0 at pH 5.8. M
(from seq) = 96577
The free tRNA sediments at 4.0 at either pH. M (from sequence) = 25744
The cognate complex sediments at about 9.0 at the lower pH (a non-cognate
tRNA does not form a complex with the enzyme) At pH 7.4 the tRNA population
essentially comigrates with the position of the free enzyme at s*=6, and we
suspect that the tRNA may be in rapid exchange

Question 1: Can I reliably determine stoichiometry by quantifying peak
areas for varying input ratios of enzyme and tRNA?  (This approach seems
similar to Preston Hensley's Fab work)

In a matched set of sed eq experiments at fixed enzyme and only two
different tRNA concentrations I collected data at three rotor speeds using
absorbance at 280 nm. This effectively minimizes the detection of the
enzyme and maximizes tRNA signal; I hoped to exploit this as a possible
means of simplifying interpretation to two species -- free or complexed
tRNA.  So far I have been using best guesses for v bar and rho.  I get
reasonable initial curve fits using the ideal model, but I'm not clear on
how to proceed for nonideal or complex association models.

Question 2: How might I approach modeling this scenario using Nonlin?

Thanks in advance for any suggestions!

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Michael L. Bovee, Ph.D.
Postdoctoral Fellow
University of Vermont
Department of Biochemistry
C444 Given Building
Burlington, VT  05405-0068
email: mbovee@zoo.uvm.edu
www: mbovee@salus.med.uvm.edu
Lab   802-656-8657
FAX  802-862-8229
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^