Calmodulin and Calmodulin-Peptide Interactions: Structure, Energetics, and Dynamics
Ehrhardt, Mark Reid
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/84889
Description
Title
Calmodulin and Calmodulin-Peptide Interactions: Structure, Energetics, and Dynamics
Author(s)
Ehrhardt, Mark Reid
Issue Date
1997
Doctoral Committee Chair(s)
Dissertation Abstracts International, Volume
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Animal Physiology
Language
eng
Abstract
Calmodulin (CaM) is the primary transducer of calcium-mediated signal transduction in many eucaryotic cells. Understanding the interactions it makes with target proteins is central to understanding its role as a master regulator of cellular metabolism. Calmodulin is capable of binding dozens of target sequences, that share little sequence similarity, with high affinity and selectivity. To explore this behavior, two high affinity peptide targets were synthesized with all D-amino acids and were found to bind calmodulin with similar affinity to their natural L counterparts. $\sp{15}$N-HSQC NMR spectra, however, revealed substantially different structural effects on CaM upon binding of D and L-smMLCK$\rm\sb{p}$ including a hundred-fold higher rate constant for dissociation of the D-peptide from CaM than for the L-peptide. The structural dynamics and energetics of the interaction between CaM and smMLCK$\rm\sb{p}$ was investigated further by employing amide hydrogen exchange techniques. Amide hydrogen exchange rates were measured at each site in the CaM-bound peptide. Slowing factors, were determined and indicated the requirement for reorganization of the complex prior to exchange. Ca$\sp{2+}$ binding normally activates target binding by CaM, however, Ca$\sp{2+}$ free CaM (apoCaM) has been found to bind to a number of targets including the neural specific protein neuromodulin. The complex formed between apoCaM and a binding domain peptide of this protein (Neuro$\rm\sb{p})$ has been studied by fluorescence spectroscopy under high hydrostatic pressure. The 12 $\mu$M complex is extremely sensitive to salt suggesting that electrostatic interactions contribute strongly to complex formation. Application of hydrostatic pressure to 34,000 psi (2.4 kbar) red shifts the emission and reduces the residual anisotropy of the single tryptophan of Neuro$\rm\sb{p}$ but does not cause dissociation of the complex. The reorganization of the complex is characterized by a volume change of $\sim$66 mL/mol and a free energy change of 1.7 kcal/mol. The development of a new beryllium-copper/sapphire high pressure NMR cell provided the means to directly observe the effects of pressure on the complex by NMR. The NMR study demonstrated that the reorganization of the apoCaM:Neuro$\rm\sb{p}$ complex was caused by the disruption of two intermolecular salt links between arginines 7 & 12 of Neuro$\rm\sb{p}$ and acidic residues on CaM.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
