Crystallization of P2 myelin protein

Bergfors, Terese; Sędzik, Jan; Unge, Torsten; Fridborg, K.; Ta, Jones; Weise, Michael

doi:10.1016/0022-2836(87)90319-6

Cited by 9 publications

(5 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Various strategies have been employed to aid in the crystallization and determination of challenging protein structures (Bergfors, 2022; Rhodes, 2006). One such approach involves fusing the protein of interest with crystallization chaperones, such as antibodies, lysozyme, glutathione S‐transferase (GST), MBP, and other highly soluble and stable proteins.…”

Section: Resultsmentioning

confidence: 99%

Structural studies of intrinsically disordered MLL‐fusion protein AF9 in complex with peptidomimetic inhibitors

Yang,

Ahmad,

Premkumar

et al. 2024

Protein Science

View full text Add to dashboard Cite

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL‐rearranged leukemias. The oncofusion proteins MLL‐AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C‐terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof‐of‐concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction‐quality crystals of the two disulfide‐bridged MBP–AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1–2.6 Å crystal complex structures provide high‐resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β‐hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein–inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

show abstract

Section: Resultsmentioning

confidence: 99%

Structural studies of intrinsically disordered MLL‐fusion protein AF9 in complex with peptidomimetic inhibitors

Yang,

Ahmad,

Premkumar

et al. 2024

Protein Science

View full text Add to dashboard Cite

show abstract

“…Mesophase for in meso crystallization of Lnt and AlgE was prepared by combining 3 volumes of dry molten 7.10 MAG with 2 volumes of protein solution. 51 For A2aR, the 7.10 MAG was first doped with 10% (w/w) cholesterol and the mesophase prepared as for Lnt and AlgE. Lnt at 13 mg/mL in Buffer A, AlgE at 35 mg/mL in Buffer B, and A2aR at 60 mg/mL in Buffer C were used for protein-laden mesophase preparation with a coupled syringe-mixing device.…”

Section: Sample Preparationmentioning

confidence: 99%

7.10 MAG. A Novel Host Monoacylglyceride for In Meso (Lipid Cubic Phase) Crystallization of Membrane Proteins

Krawinski,

Smithers,

van Dalsen

et al. 2024

Crystal Growth & Design

View full text Add to dashboard Cite

A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature−composition phase diagrams for 7.10 MAG have been constructed using small-and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG's chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid.

show abstract

“…The phase behavior study, nevertheless, showed that crystalline precipitates could grow out of the heavy precipitates suggesting that the Van molecules in the heavy precipitates remained in their native state, rather than unfolding. Furthermore, the heavy precipitates were found to readily redissolved in water, which would have not been possible if the Van precipitates had denatured [321]. As we discussed in Section 3.4, further characterizations by PXRD and FTIR confirmed the structural integrity of the heavy precipitates.…”

Section: Phase Behavior Of Antisolvent Van Precipitationmentioning

confidence: 74%

“…The phase behaviors of antisolvent Van precipitation were characterized using the classification method for protein precipitation developed by Bergfors [321].…”

Section: Phase Behavior Of Antisolvent Van Precipitationmentioning

confidence: 99%

Advanced strategies to enhance the performances of crystallization and precipitation of biopharmaceuticals

Pu¹

View full text Add to dashboard Cite

Recent years have seen significant growth in the production titer of upstream of biopharmaceuticals, a level that could lead to diminishing returns upon increasing due to the downstream processing (DSP) bottleneck. The capture and polishing steps, representing the most laborious and expensive aspects of the DSP, are currently mainly dominated by pack-bed chromatography. However, due to the high cost of adsorbent and the high operation expenditure of chromatography-based theology, anything but chromatography (ABC) methodologies started to gain interest in the field as alternative method for purifying biopharmaceuticals. Precipitation and crystallization are classic unit operations that can provide promising alternatives in DSP. The aim of this dissertation is to develop advanced crystallization and precipitation strategies to improve process efficiency and product quality of biopharmaceuticals using bioactive Van glycopeptide and protein LYZ as the model biopharmaceuticals In the first study, we investigated the phase behavior of salting-out roomtemperature crystallization of glycopeptide antibiotics to determine crystallization conditions (i.e., pH, salt and peptide's concentrations, incubation time) to avoid needle-shaped crystal formation. Batch crystallizations of the prominent crystal habits identified from the phase behavior study (i.e., needle and non-needle) were subsequently performed. The batch crystallization's products were evaluated in their production yield and capacity, purity, size distribution, thermal stability, interfacial water content, dissolution profile, and antibiotic activity. The results showed that octahedral crystals were the predominant habit produced across the range of pH, salt, and peptide concentrations investigated. Needle crystal formation could be avoided by precise controls of pH and salt concentration. Octahedral crystals were produced at a significantly higher yield than needle crystals. The octahedral and needle crystals exhibited many similar properties but with distinct thermal stability and dissolution profiles.The second study compared the antisolvent (with organic solvent) and salting-out methods in their precipitation efficiency and product qualities. The phase behavior study showed that heavy precipitates composed of nanoparticles were the

show abstract

Crystallization of P2 myelin protein

Cited by 9 publications

References 19 publications

Structural studies of intrinsically disordered MLL‐fusion protein AF9 in complex with peptidomimetic inhibitors

Structural studies of intrinsically disordered MLL‐fusion protein AF9 in complex with peptidomimetic inhibitors

7.10 MAG. A Novel Host Monoacylglyceride for In Meso (Lipid Cubic Phase) Crystallization of Membrane Proteins

Advanced strategies to enhance the performances of crystallization and precipitation of biopharmaceuticals

Contact Info

Product

Resources

About