Monitoring calcium-induced conformational changes in recoverin by electrospray mass spectrometry.
Recoverin is a calcium-binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N-acylated by myristoyl and related fatty acyl residues that are thought to act as "calcium-myristoyl switches," whereby, in the presence of Ca2+, the N-terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely associated with the protein. Here we use electrospray ionization mass spectrometry (ESI/MS) to examine hydrogen isotopic exchange rates for specific regions of both acylated and nonacylated recoverin in the presence and absence of calcium. The deuterium exchange rates of three regions in the hydrophobic myristoyl binding pocket of acylated recoverin decreased in the absence of calcium. This effect is most likely due to the closer association of the acyl group with the protein under these conditions. In contrast, rates of deuterium incorporation increased in the absence of calcium for other regions, including the two functional calcium-binding sites. In addition to supporting the calcium-myristoyl switch hypothesis, a comparison of the behavior of acylated and unacylated recoverin revealed that the N-acyl group (N-lauroyl or N-myristoyl) exerts a significant stabilizing influence on the dynamics of recoverin. We demonstrate that the new technique of monitoring hydrogen isotopic exchange by ESI/MS can be used to obtain useful information concerning protein structures in solution using smaller amounts of protein and under more physiologically relevant conditions than is typically possible with NMR or X-ray crystallography.
Neubert
T A
TA
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Walsh
K A
KA
Hurley
J B
JB
Johnson
R S
RS
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D051603:Recoverin
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium