您的位置 首页 工具使用

通过计算机设计来降低抗体片段构象的灵活性及聚合速率

嘉宾:章程博士,伦敦大学学院
主持人:惠成功
时间:2019年9月3日晚20:00

摘要:
Computationally guided semirational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilize proteins to aggregation under some conditions, previous studies suggest that such an approach is limited, because thermal transition temperatures ( Tm) and the fraction of protein unfolded ( fT) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the Tm. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, which reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilizing mutations to regions of the A33 Fab surface structure, which were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔGND for each potential point mutation. Additional destabilizing variants were prepared as controls to evaluate the prediction accuracy and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of 18 variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased Tm values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by Tm. However, 6 of the 12 stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate.
KEY WORDS:
Fab; aggregation; cooperativity; entropy; global unfolding; melting temperature (Tm); molecular dynamics; mutagenesis; protein engineering; thermal stabilit



发表回复

您的电子邮箱地址不会被公开。 必填项已用*标注

评论列表(91)

  1. Link pyramid, tier 1, tier 2, tier 3
    Top – 500 connections with inclusion contained in writings on writing sites

    Secondary – 3000 link Forwarded links

    Tertiary – 20000 hyperlinks combination, posts, articles

    Using a link hierarchy is advantageous for indexing systems.

    Need:

    One hyperlink to the platform.

    Search Terms.

    Correct when 1 keyword from the content heading.

    Observe the supplementary feature!

    Essential! Tier 1 connections do not conflict with Tier 2 and 3rd-tier links

    A link structure is a tool for boosting the movement and link profile of a website or virtual network

  2. Wow, incredible weblog layout! How lengthy have you ever been blogging for?
    you made running a blog glance easy. The total glance of your
    website is magnificent, let alone the content! You can see similar
    here dobry sklep

联系我们

联系我们

(44)07934433023

在线咨询: QQ交谈

邮箱: info@bioengx.org

关注微信
微信扫一扫关注我们

微信扫一扫关注我们

关注微博
返回顶部