Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases

Tovar-Anaya, David O.; Vera-Robles, L. Iraís; Vieyra-Eusebio, María Teresa; García-Gutiérrez, Ponciano; Reyes-Espinosa, Francisco; Hernández‐Arana, Andrés; Arroyo-Reyna, J. Alfonso; Zubillaga, Rafael A.

doi:10.1007/s10930-019-09839-2

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“…The heterologous HCC expression was carried out in SHuffle ® T7-competent E. coli cells (New England Biolabs, Ipswich, MA, USA) using a modified protocol [ 34 ]. The bacteria were grown in an LB standard medium (10 g/L of tryptone, 5 g/L of yeast extract and 10 g/L of NaCl) (BioShop Canada, Burlington, ON, Canada) with kanamycin (50 µg/mL) ((BioShop Canada) at 30 °C, with shaking at 180 rpm (Lab Companion, ISS-4075R).…”

Section: Methodsmentioning

confidence: 99%

Oligomerization of Human Cystatin C—An Amyloidogenic Protein: An Analysis of Small Oligomeric Subspecies

Wojciechowska

Taube

Rucińska

et al. 2022

IJMS

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Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0–5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4.

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Section: Methodsmentioning

confidence: 99%