Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1

Chandra, Kousik; Sharma, Yogendra; Chary, Kandala V. R.

doi:10.1016/j.bbapap.2010.10.007

Cited by 8 publications

(15 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An interesting feature of this peptide-binding site is that it overlaps with the myristoyl-binding pocket identified in the NCS homolog GCAP-1 (39). Like NCS-1 (40), the myristoyl moiety is not involved in a calcium-myristoyl switch, and thus interacts with the protein in the Ca 2+ free state. This would provide an explanation for the reduced affinity of Myr-NSC-1 for the D2R peptide, since it would have to compete with the myristoyl-moiety for binding.…”

Section: Resultsmentioning

confidence: 99%

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

et al. 2011

View full text Add to dashboard Cite

Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF hand variants to interrogate the interaction between the D2R and NCS-1. Our data is consistent with the following conclusions: 1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data is best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. 2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. 3) N-terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. 4) Co-immunoprecipitation experiments in HEK293 confirm that NCS-1 can oligomerize in cell lysates, and that oligomerization is dependent on calcium binding and requires functionally intact EF hand domains. 5) Ca2+/Mg2+ FA titration experiments revealed that NCS-1 EF-hands 2-4 contributed to binding with the D2R peptide. EF-2 appears to have the highest affinity for Ca2+ and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological co-factor with calcium for NCS-1/D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1.

show abstract

Section: Resultsmentioning

confidence: 99%

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

et al. 2011

View full text Add to dashboard Cite

show abstract

“…The dynamics of these conformations are central to protein stability, binding, allostery, and catalysis in ways that remain difficult to explain and predict. − Protein dynamics are challenging to characterize as they are invisible to many standard techniques and occur on many different time scales with varying energetics. Measurement of the temperature-dependence of chemical shifts by nuclear magnetic resonance (NMR) is a powerful approach that can provide high resolution information on both local structure and conformational heterogeneity in proteins. − In particular, the linear dependence of amide proton chemical shifts (δ NH ) on temperature, known as the temperature coefficient, Δδ NH /Δ T , is well established as being sensitive to hydrogen bond formation. ,,,,, However, recent work has found that a larger determinant of Δδ NH /Δ T is the temperature-dependent loss of structure and that the average and spread of Δδ NH /Δ T values reports on structure even in the absence of persistent intramolecular hydrogen bonding. , Here, we explore further how Δδ NH /Δ T can be used to characterize temperature-dependent loss of structure within proteins, which we refer to as “structural stability.”…”

mentioning

confidence: 99%

“…In addition, the nonlinearity of the temperature-dependence of δ NH can report on the population of alternative states of proteins, i.e., “conformational heterogeneity.” Previous studies of various proteins have found that between 5% and 40% of amide protons exhibit curved temperature-dependences. ,,,,, The curvature was modeled in terms of the population of an alternative state in fast exchange on the chemical shift time scale with the ground state and within ∼5 kcal/mol in energy. , Thus, measurements of curvature can report on alternative states close in energy to the native state and may complement other NMR methods for identifying and structurally characterizing thermally accessible low lying biomolecular conformations. − To date, comparisons of the temperature-dependences of δ NH for related proteins have been limited, for example, to characterizing similarities and differences between homologous proteins from different organisms or caused by myristoylation. , …”

mentioning

confidence: 99%

“…6 Previous studies of various proteins have found that between 5% and 40% of amide protons exhibit curved temperature-dependences. 6,7,10,17,19,20 The curvature was modeled in terms of the population of an alternative state in fast exchange on the chemical shift time scale with the ground state and within ∼5 kcal/mol in energy. 6,20 Thus, measurements of curvature can report on alternative states close in energy to the native state and may complement other NMR methods for identifying and structurally characterizing thermally accessible low lying biomolecular conformations.…”

mentioning

confidence: 99%

“…26−32 To date, comparisons of the temperature-dependences of δ NH for related proteins have been limited, for example, to characterizing similarities and differences between homologous proteins from different organisms or caused by myristoylation. 10,19 Here, we apply measurements of Δδ NH /ΔT, as well as nitrogen chemical shift temperature-dependence (Δδ N /ΔT) which have been less studied, and curvature of δ NH to investigate changes caused by maturation and disease-associated mutations for human Cu, Zn superoxide dismutase (SOD1). This homodimeric metalloenzyme consists of 153 amino acid subunits, each binding 1 Cu 2+ and 1 Zn 2+ , containing a conserved intrasubunit disulfide bond and adopting an immunoglobulinlike antiparallel β-barrel fold with an N-terminal sheet (strands 1, 2, 3, and 6) and a C-terminal sheet (strands 4, 5, 7, and 8).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts

Doyle¹,

Rumfeldt²,

Broom³

et al. 2016

Biochemistry

View full text Add to dashboard Cite

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

show abstract

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis

et al. 2019

View full text Add to dashboard Cite

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

show abstract

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1

Cited by 8 publications

References 62 publications

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis

Contact Info

Product

Resources

About