X-ray crystallography has tremendously served structural biology by routinely providing high-resolution 3D structures of macromolecules. The extent of information encoded in the X-ray crystallography is proportional to which resolution the crystals diffract and the structure can be refined to. Therefore, there is a continuous effort to obtain high-quality crystals, especially for those proteins, which are considered difficult to crystallize into high-quality protein crystals of suitable sizes for X-ray crystallography. Efforts in enhancing the resolution in X-ray crystallography have also been made by optimizing crystallization protocols using external stimuli such as an electric field and magnetic field during the crystallization. Here, we present the feasibility of on-the-fly post-crystallization resolution enhancement of the protein crystal diffraction by applying a high-voltage electric field. The electric field between 2 and 11 kV/cm, which was applied after mounting the crystals in the beamline, resulted in the enhancement of the resolution. The crystal diffraction quality improved progressively with the exposure time. Moreover, we also find that upto defined electric field threshold, the protein structure remains largely unperturbed, a conclusion further supported by molecular dynamics simulations.

• Keywords
• External electric field, Macromolecular crystals, Resolution enhancement,
• MeSH
• Electricity * MeSH
• Crystallography, X-Ray methods   MeSH
• Proteins * chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteins * MeSH

Hemoglobin is an oxygen-transport protein in red blood cells that interacts with multiple ligands, e.g., oxygen, carbon dioxide, carbon monoxide, and nitric oxide. Genetic variations in hemoglobin chains, such as those underlying sickle cell disease and thalassemias, present substantial clinical challenges. Here, we review the progress in research, including the use of allosteric modulators, pharmacological chaperones, and antioxidant treatments, which has begun to improve hemoglobin stability and oxygen affinity. According to UniProt (as of 7 August 2024), 819 variants of the α-hemoglobin subunit and 771 variants of the β-hemoglobin subunit have been documented, with over 116 classified as unstable. These data demonstrate the urgent need to develop variant-specific stabilizing options. Beyond small-molecule drugs/binders, novel protein-based strategies-such as engineered hemoglobin-binding proteins (including falcilysin, llama-derived nanobodies, and α-hemoglobin-stabilizing proteins)-offer promising new options. As our understanding of hemoglobin's structural and functional diversity grows, so does the potential for genotype-driven approaches. Continued research into hemoglobin stabilization and ligand-binding modification may yield more precise, effective treatments and pave the way toward effective strategies for hemoglobinopathies.

• Keywords
• 2,3-bisphosphoglycerate (2,3-BPG), Bohr effect, allosteric regulation, genetic variants, hemoglobin (Hb), oxygen affinity, oxygen-binding properties, protein engineering, sickle cell disease (SCD), thalassemia,
• MeSH
• Hemoglobinopathies drug therapy   genetics   metabolism   MeSH
• Hemoglobins * genetics   chemistry   metabolism   MeSH
• Humans MeSH
• Protein Stability drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Hemoglobins * MeSH