Non-Additive Effects of Binding Site Mutations in Calmodulin

Abstract

Despite decades of research on ion-sensing proteins, gaps persist in the understanding of ion binding affinity and selectivity even in well-studied proteins such as calmodulin. Site-directed mutagenesis is a powerful and popular tool for addressing outstanding questions about biological ion binding and is employed to selectively deactivate binding sites and insert chromophores at advantageous positions within ion binding structures. However, even apparently nonperturbative mutations can distort the binding dynamics they are employed to measure. We use Fourier transform infrared (FTIR) and ultrafast two-dimensional infrared (2D IR) spectroscopy of the carboxylate asymmetric stretching mode in calmodulin as a mutation-and label-independent probe of the conformational perturbations induced in calmodulin's binding sites by two classes of mutation, tryptophan insertion and carboxylate side-chain deletion, commonly used to study ion binding in proteins. Our results show that these mutations not only affect ion binding but also induce changes in calmodulin's conformational landscape along coordinates not probed by vibrational spectroscopy, remaining invisible without additional perturbation of binding site structure. Comparison of FTIR line shapes with 2D IR diagonal slices provides a clear example of how nonlinear spectroscopy produces well-resolved line shapes, refining otherwise featureless spectral envelopes into more informative vibrational spectra of proteins.

Probing Lanmodulin’s Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage

“…As a result of the substantial perturbation introduced by the Trp residue in EF2, this site was not directly probed further, although T90W in EF3 does appear to report on metal binding to EF2 as well (vide infra). Disruptive effects resulting from substitution of Trp residues at the EF-hand seventh position have been observed previously for some Ca II binding EF-hand proteins . The other variants displayed full conformational response to 3 equiv of Tb III with the exception of N87W and T90W, which required 4 equiv of Tb III , similar to the fluorescence result (Figure S2).…”

“…Disruptive effects resulting from substitution of Trp residues at the EF-hand seventh position have been observed previously for some Ca II binding EF-hand proteins. 52 The other variants displayed full conformational response to 3 equiv of Tb III with the exception of N87W and T90W, which required 4 equiv of Tb III , similar to the fluorescence result (Figure S2). K d,app values were determined for the Tb III -bound Trp variants in comparison to wt LanM (Figure 2, Table 1).…”

The Chemistry of Lanthanides in Biology: Recent Discoveries, Emerging Principles, and Technological Applications

“…Studies have suggested positive impacts of RE provision in animal husbandry, suggesting a role for REs in higher organisms. 128 However, REs are not perfect mimics of Ca, 4 as recent studies of calmodulin 129,130 and cadherin 131 have shown, suggesting that REs may not play a general role as Ca substitutes in Ca-dependent proteins. Instead, the growth-promoting effects of REs in higher organisms might plausibly be linked to the microbiome; for example, both beneficial ( E. coli ) and pathogenic ( Pseudomonas aeruginosa ) bacteria possess PQQ-dependent dehydrogenases, and these or other enzymes might benefit from or be inhibited by REs.…”

CH Mode Mixing Determines the Band Shape of the Carboxylate Symmetric Stretch in Apo-EDTA, Ca2+–EDTA, and Mg2+–EDTA

“…17 Separate active site mutations in CaM show different antisymmetric stretch cross-peak structures, which are the manifestations of the CaM binding pocket's conformational flexibility. 19 In each of these examples, the COO a − bands in 2D-IR spectra provided important insights into local structure and dynamics of these carboxylate moieties.…”

“…Though EDTA mimics the EF-hand binding pocket, spectroscopic studies fail to unambiguously correlate metal binding geometries with the positions and intensities of the carboxylate vibrational bands. − The additional information content of two-dimensional infrared (2D-IR) spectroscopy promises to allow more direct relationships between observed spectral features and ion-binding geometries. Additionally, it can provide insight into the femtosecond and picosecond dynamics in the binding pocket. − …”