Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

2D IR Correlation Spectroscopy in the Determination of Aggregation and Stability of KH Domain GXXG Loop Peptide in the Presence and Absence of Trifluoroacetate.

Trifluoroacetate (TFA) is a strong anion byproduct of solid-phase peptide synthesis. Fourier transform infrared (FT-IR) spectroscopy can be used to ascertain the presence of this excipient in peptide samples for quality assessment. TFA absorbs as a strong sharp peak (1675 cm-1) within the amide I' band of the spectral region. A peptide sample and the TFA excipient can be studied simultaneously by FT-IR and 2D IR correlation spectroscopies. In addition, these techniques are able to determine the effect of TFA on the stability of the peptide. Herein, we describe the spectroscopic characterization of the GXXG loop peptide (GXXGlp), which is present in KH domain containing proteins. The sequence of the Homo sapiens Krr1 GXXGlp is evolutionarily conserved (165KRRQRLIGPKGSTLKALELLTNCY189) and has been associated with ssDNA interaction and ribosome biogenesis. Our goal was to determine the structural elements present in this peptide and evaluate whether TFA affects the stability of GXXGlp during thermal stress. We observed differences in the molecular behavior of the synthetic peptide in the presence and absence of TFA at various peptide concentrations. Finally, 2D IR correlation spectroscopy was used for the determination of the unfolding process, mechanism and extent of peptide aggregation, and the effect of TFA on the stability of the peptide. This spectroscopic method can be applied to the characterization of any synthetic peptide.

Relative stability of human centrins and its relationship to calcium binding.

Centrins are calcium binding proteins that belong to the EF-hand superfamily with diverse biological functions. Herein we present the first systematic study that establishes the relative stability of related centrins via complementary biophysical techniques. Our results define the stepwise molecular behavior of human centrins by two-dimensional infrared (2D IR) correlation spectroscopy, the change in heat capacity and enthalpy of denaturation by differential scanning calorimetry, and the relative stability of the helical regions of centrins by circular dichroism. More importantly, 2D IR correlation spectroscopy provides unique information about the similarities and differences in dynamics between these related proteins. The thermally induced molecular behavior of human centrins can be used to predict biological target interactions that have a relative dependence on calcium affinity. This information is essential for understanding why certain isoforms may be used to rescue a phenotype and therefore also for explaining the different functions these proteins may have in vivo. Furthermore, this comparative approach can be applied to the study of recombinant therapeutic protein candidates for the treatment of disease states.