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The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone-client complexes . The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA.

Background: High altitude renal syndrome has been described in populations with excessive erythrocytosis. We evaluated whether high altitude (HA) dwellers might be at increased risk for kidney disease., Methods: We performed a cross-sectional study to investigate differences in prevalence of kidney function and metabolic syndrome in healthy subjects living at HA vs. sea level (SL) without any known history of hypertension, diabetes or chronic kidney disease., Results: We examined 293 subjects, aged 40 to 60 years: 125 SL (154 m) and 168 HA (3640 m) dwellers. HA dwellers had higher serum creatinine, lower estimated glomerular function rate (eGFR) (69.5 ± 15.2 vs. 102.1 ± 17.8 ml/min/1.73 m 2 , p < 0.0001), more proteinuria and higher hemoglobin concentrations compared to SL subjects. HA subjects had a lower prevalence of metabolic syndrome. Hemoglobin concentrations correlated inversely with eGFR in female (p = 0.001) and male (p = 0.03) HA dwellers. Using logistic regression analysis to compare subjects with eGFR < 90 vs. > 90 ml/min/1.73 m 2 , a lower eGFR was associated with female gender (odds ratio adjusted: 5.65 [95% confidence interval: 2.43-13.13]; p = 0.001), high altitude (14.78 [6.46-33.79]; p = 0.001), hemoglobin (1.68 [1.16-2.43]; p = 0.001) and uric acid (1.93 [1.36-2.72]; p = 0.001)., Conclusions: Dwellers at high altitude who are considered healthy have worse kidney function, a higher prevalence of proteinuria and a lower prevalence of metabolic syndrome compared to people living at SL.

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.