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Title:Calmodulin and Calmodulin-Peptide Interactions: Structure, Energetics, and Dynamics
Author(s):Ehrhardt, Mark Reid
Doctoral Committee Chair(s):Dissertation Abstracts International, Volume
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Animal Physiology
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.
Issue Date:1997
Description:191 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.
Other Identifier(s):(MiAaPQ)AAI9834669
Date Available in IDEALS:2015-09-25
Date Deposited:1997

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