From: John Correia <jcorreia@biochem.umsmed.edu>
  To  : rasmb@alpha.bbri.org
  Date: Wed, 27 Jan 1999 14:01:41 -0600

His tags

I sent this message to Richard earlier & didn't send it to rasmb in
general; here it is with an addendum added after I read John Philo's
comments:

Richard,

I worked on two his tagged kinesin constructs, K332 and K413, that
exhibited aggregation phenomena and required colder temperatures to
suppress it, not remove it, and the use of sed vel s/D measurements to
verify the MW's as monomer.  I specifically say 'maybe' its due to the his
tags.  The caveat is we never checked the nonhis tagged constructs - Steve
had trouble purifying them without the tag and so we never did the direct
test.  This is a significant caveat because many of these kinesin
constructs exhibit aggregation; we're currently working with K420 and it
is also is prone to aggregation.  In my mind K332, the monomer, was the
best example because other monomers, K341 & K366, I worked on earlier with
Susan Gilbert did not exhibit aggregation.

See Rosenfeld, S.S., J.J. CORREIA, J. Xing, Z. Gong, B. Rener and H.C.
Cheung (1996)  "Structural Studies of Kinesin:Nucleotide Intermediates",
J. Biol. Chem., 271, 30212-30221.

CORREIA,J.J., S.P. Gilbert, M.L. Moyer and K.A. Johnson (1995)  
"Sedimentation Studies on The Kinesin Head Domain Constructs K401, K366 and K341. 
Biochemistry, 34, 4898-4907.

addendum:  What is the mechanism of his tag induced aggregation?  John
suggests they directly interact with the rest of the protein, maybe
disrupting structure and inducing ?hydrophobic patches or something that
aggregate?  Is there any evidence that the his tag itself has affinity for
another his tag??  In many kinesin constructs the tag is usually at the
end of a coiled coil domain so looping back to interact with the motor
domain or a globular domain is unlikely.


-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
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