Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Abstract

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca 2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca 2+ -ATPase.Single-molecule fluorescence methods have the potential to resolve details about protein conformations, interactions, and dynamics that were previously hidden by ensemble averaging. As the practice of single-molecule methods expands, it is useful to examine what new information can be obtained about biomolecules and what the limitations of the methods are. This paper will examine these questions in the context of recent applications of single-molecule fluorescence techniques in the author's laboratory to the conformations, dynamics, and interactions of the calcium signaling protein calmodulin (CaM) (see Fig. 1).Proteins are dynamic molecules. Multiple conformational states are required for them to function. CaM is no exception, and appears to sample an unusually wide range of conformations (1). How can the distribution of such conformations be characterized? Conventional bulk measurements detect the average properties of many (typically > 10 10 ) molecules and therefore do not resolve the range of conformations that are actually present. One way to attack this problem is to detect and characterize molecules one at a time.Introduced in the early 1990s (2-6), single-molecule fluorescence methods can now address real problems in biology (see recent reviews (7-10)). Conformational states and conformational motions can be detected that may be missed by other methods. Dynamic processes can be probed under either equilibrium or non-equilibrium conditions by following the fluctuations of an appropriate single-molecule spectroscopic signal. In kinetic *To whom correspondence should be addressed. E-mail: ckjohnson@ku.edu. † We acknowledge support for this research from NIH R01 GM58715, the American Heart Association (99513262 and 0455487Z) and a Research Corporation Research Opportunity Award. B.D.S. and J.R.U. acknowledge support from training grant NIH GM08545, and E.S.P. acknowledges support from t...

Dynamic personalities of proteins

“…5c). This is supported by several lines of evidence: NMR data indicate that the linker helix between the domains is flexible in the absence of peptide 75 ; X-ray diffraction has trapped both an extended structure (Protein Data Bank (PDB) identity 1CLL) 76 and a closed structure (PDB identity 1PRW) 77 ; and single-molecule FRET distributions show that a wide range of interdomain distances are sampled 78,79 . This flexibility of calmodulin on the microsecond-to-millisecond timescale allows the ligands to select their preferred conformation, explaining the specificity of calmodulin for so many targets.…”

“…Signal transduction, enzyme catalysis and protein-ligand interactions occur as a result of the binding of specific ligands to complementary pre-existing states of a protein and the consequent shifts in the equilibria 26,35,36,[75][76][77][78][79][90][91][92] (Fig. 1a).…”

The Complex Inter-Relationships Between Protein Flexibility and Stability

“…[140][141][142][143][144][145] Numerous studies have shown that the apo form of CaM is much less stable than the Ca 2þ bound form. 141,144,145 Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) among many other methods have demonstrated decreased conformational dynamics and compaction of CaM upon Ca 2þ binding.…”

Entropic mechanism of large fluctuation in allosteric transition

“…Recent experimental data have shown the existence of fluctuations among multiple native conformations prior to the effector binding in several example allosteric proteins such as NtrC (nitrogen regulatory protein C) (7,8), CheY (9)(10)(11), calmodulin (12)(13)(14), and adenylate kinase (15)(16)(17), which has supported the view that the population-shift mechanism is not exceptional but may be ubiquitous in allosteric transitions (18)(19)(20)(21)(22)(23). There still remain, however, important questions to be elucidated: First, the actual mechanism in each allosteric protein may reside between two limits of population shift and induced fit, so that the relative importance of two mechanisms should be analyzed in each case (24).…”