Effective scavenging at cryotemperatures: further increasing the dose tolerance of protein crystals.

J Synchrotron Radiat

Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Published: May 2011

The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.5 M in chicken egg-white lysozyme crystals, the dose tolerance is increased by a factor of two as judged from the global damage parameters, and no specific structural damage to the disulfide bonds is seen until the dose is greatly in excess (more than a factor of five) of the value at which damage appears in electron density maps derived from a scavenger-free crystal. In the electron density maps, ordered nitrate ions adjacent to the disulfide bonds are seen to lose an O atom, and appear to protect the disulfide bonds. In addition, results reinforcing previous reports on the effectiveness of ascorbate are presented. The mechanisms of action of both scavengers in the crystalline environment are elucidated.

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0909049511007163DOI Listing

Publication Analysis

Top Keywords

disulfide bonds
12
dose tolerance
8
sodium nitrate
8
electron density
8
density maps
8
effective scavenging
4
scavenging cryotemperatures
4
cryotemperatures further increasing
4
further increasing dose
4
tolerance protein
4

Similar Publications

A motion-responsive injectable lubricative hydrogel for efficient Achilles tendon adhesion prevention.

Mater Today Bio

February 2025

Basic Research Key Laboratory of General Surgery for Digital Medicine, Affiliated Hospital of Hebei University, Baoding, 071000, China.

Achilles tendon is a motor organ that is prone to tissue adhesion during its repair process after rupture. Therefore, developing motion-responsive and anti-adhesive biomaterials is an important need for the repair of Achilles tendon rupture. Here, we report an injectable lubricative hydrogel (ILH) based on hydration lubrication mechanism, which is also motion-responsive based on sol-gel reversible transmission.

View Article and Find Full Text PDF

Platelet factor 4 (PF4), a specific protein primarily found in megakaryocytes and platelet α-granules, plays an essential role in the coagulation process. It carries a high positive charge and thus has a unique ability to readily form complexes with negatively charged heparin. This interaction between PF4 and heparin plays a crucial role in platelet aggregation and thrombosis, resulting in heparin-induced thrombocytopenia (HIT).

View Article and Find Full Text PDF

The production of disulfide-containing recombinant proteins often requires refolding of inclusion bodies before purification. A pre-refolding purification step is crucial for effective refolding because impurities in the inclusion bodies interfere with refolding and subsequent purification. This study presents a new pre-refolding procedure using a reversible S-cationization technique for protein solubilization and purification by reversed-phase high performance liquid chromatography.

View Article and Find Full Text PDF

Light chain Split Luciferase assay implicates pathological NOTCH3 thiol reactivity in inherited cerebral small vessel disease.

J Biol Chem

January 2025

Departments of Neurology, University of Michigan, Ann Arbor, MI 48109; Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109; Neurology Service, VA Ann Arbor Healthcare System, Department of Veterans Affairs, Ann Arbor, MI 48105. Electronic address:

Stereotyped mutations in NOTCH3 drive CADASIL, the leading inherited cause of stroke and vascular dementia. The vast majority of these mutations result in alterations in the number of cysteines in the gene product. However, non-cysteine altering pathogenic mutations have also been identified, making it challenging to discriminate pathogenic from benign NOTCH3 sequence variants.

View Article and Find Full Text PDF

Dectin-1 (CLEC7A), a C-type lectin-like receptor that recognizes β-1,3 glucans, has a key role in the innate immune system. While the lectin domain of mouse Dectin-1 has been solubilized and refolded from inclusion bodies in Escherichia coli, similar refolding of the human Dectin-1 lectin domain is hindered by the formation of misfolded multimers with aberrant intermolecular disulfide bonds. The aim of this study was to develop a method for the large-scale production of the human Dectin-1 lectin domain.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!