Thermodynamics of binding by calmodulin correlates with target peptide α‐helical propensity

Dunlap, Tori B.; Kirk, Jessime M.; Pena, Emily A.; Yoder, Meghan S.; Creamer, T.

doi:10.1002/prot.24215

Cited by 24 publications

(24 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Positive, neutral and negative temperature-induced rate changes all occur, indicating that the EF-hand and peptide mutations have sensitively affected the balance of entropy and enthalpy gains. Favourable dehydration of hydrophobic surfaces results in entropy-driven activation and a positive temperature coefficient30. The lack of temperature dependence of the rates, e.g.…”

Section: Discussionmentioning

confidence: 99%

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Helassa

Podor

Fine

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

show abstract

Section: Discussionmentioning

confidence: 99%

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Helassa

Podor

Fine

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Lastly, using CD methods similar to those used previously to analyze coupled folding and binding of calmodulin binding targets to calmodulin; we directly confirmed the structural transition of the BH3D by assessing and comparing the secondary structure content (Fig. and Table ) of different molar mixtures of BCL2 and the BECN 1 BH3D, as well as of BCL2 and DS BH3D, which does not bind to BCL2 (Table ) and is also disordered in solution (Fig.…”

Section: Resultsmentioning

confidence: 57%

Intrinsically disordered regions in autophagy proteins

et al. 2013

View full text Add to dashboard Cite

Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein-protein interactions, and delineate the network of proteins that interact with each predicted IDR-containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α-helix upon binding to BCL2s, with the C-terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in-depth investigations of the biological role and mechanism of IDRs in autophagy proteins.

show abstract

“…JPred4 [22] was used to predict potential secondary structure. We used circular dichroism (CD) spectroscopy to monitor change in secondary structure content of diverse IDRs induced by 2,2,2-trifluoroethanol (TFE), which is known to stabilize α-helical structure in disordered amino acid sequences that have some native tendency to form helices [23-27]. Our results demonstrate that IDRs that are not predicted by ANCHOR to fold upon binding do not become helical in the presence of TFE.…”

Section: Introductionmentioning

confidence: 99%

Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions

Glover

Yang

Sinha

2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

View full text Add to dashboard Cite

Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions.

show abstract

Thermodynamics of binding by calmodulin correlates with target peptide α‐helical propensity

Cited by 24 publications

References 29 publications

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

Intrinsically disordered regions in autophagy proteins

Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions

Contact Info

Product

Resources

About