The overall goal of this project is to detect single calmodulin(CaM) molecules as they function. Key questions need to be answered about how CaM recognizes and binds to a target. CaM is a key link in many biochemical calcium signaling pathways. The flexible structure of CaM and the wide variety of potential targets suggests that a distribution of conformations exists at the molecular level. Single-molecule experiments are therefore proposed in order to investigate conformational fluctuations and heterogeneity of CaM and CaM-target complexes. Whereas conventional methods measure an ensemble average, single-molecule measurements probe individual variations in structure and target activation. Preliminary investigations are described that have uncovered a distribution of binding conformations for CaM bound to the CaM-binding domain of the plasma membrane Ca-ATPase (PMCA). It is suggested that this distribution correlates with a distribution of activity levels of the enzyme, providing a mechanism for fine-tuning of enzyme regulation. This proposal seeks to test this hypothesis. The proposal also seeks to understand the molecular mechanisms of target recognition and binding by detecting individual binding events. Such measurements are needed to probe targets. The proposed research will also measure the coupling between CaM-binding and enzyme activation in the PMCA, permitting a detailed investigation of this mechanism. An important step in the project will be developing methods to immobilize the protein during observation and implementing fluorescence probes of single-molecule binding and activation. Because calmodulin regulates numerous biological processes, this work is pertinent to a wide range of health concerns, including neurotransmission and learning, oxidative damage and aging, and muscle activation. An understanding of the interaction between calmodulin and target proteins under conditions involving cellular signaling or oxidative stress will be crucial in designing therapies to alleviate disorders involving these processes. The experimental approach will combine recently developed techniques of single-molecule spectroscopy with fluorescence resonance energy transfer and site-directed mutagenesis as a probe of protein structure and dynamics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058715-04
Application #
6643503
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Lewis, Catherine D
Project Start
2000-09-01
Project End
2005-08-31
Budget Start
2003-09-01
Budget End
2005-08-31
Support Year
4
Fiscal Year
2003
Total Cost
$227,344
Indirect Cost
Name
University of Kansas Lawrence
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Slaughter, Brian D; Urbauer, Ramona J Bieber; Urbauer, Jeffrey L et al. (2007) Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation. Biochemistry 46:4045-54
Johnson, Carey K (2006) Calmodulin, conformational states, and calcium signaling. A single-molecule perspective. Biochemistry 45:14233-46
Slaughter, Brian D; Bieber-Urbauer, Ramona J; Johnson, Carey K (2005) Single-molecule tracking of sub-millisecond domain motion in calmodulin. J Phys Chem B 109:12658-62
Slaughter, Brian D; Unruh, Jay R; Allen, Michael W et al. (2005) Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification. Biochemistry 44:3694-707
Slaughter, Brian D; Unruh, Jay R; Price, E Shane et al. (2005) Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy. J Am Chem Soc 127:12107-14
Johnson, Carey K; Osborn, Kenneth D; Allen, Michael W et al. (2005) Single-molecule fluorescence spectroscopy: new probes of protein function and dynamics. Physiology (Bethesda) 20:10-4
Osborn, Kenneth D; Zaidi, Asma; Urbauer, Ramona J Bieber et al. (2005) Single-molecule characterization of the dynamics of calmodulin bound to oxidatively modified plasma-membrane Ca2+-ATPase. Biochemistry 44:11074-81
Allen, Michael W; Urbauer, Ramona J Bieber; Johnson, Carey K (2004) Single-molecule assays of calmodulin target binding detected with a calmodulin energy-transfer construct. Anal Chem 76:3630-7
Osborn, Kenneth D; Zaidi, Asma; Mandal, Abhijit et al. (2004) Single-molecule dynamics of the calcium-dependent activation of plasma-membrane Ca2+-ATPase by calmodulin. Biophys J 87:1892-9
Osborn, Kenneth D; Bartlett, Ryan K; Mandal, Abhijit et al. (2004) Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin. Biochemistry 43:12937-44

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