On the analysis of sedimentation velocity in the study of protein complexes.
On the analysis of sedimentation velocity in the study of protein complexes.
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DOI：
10.1007/s00249-009-0514-1
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发表时间：
2009-10
影响因子：
2
通讯作者：
Schuck, Peter
中科院分区：
文献类型：
--
作者：
Brown, Patrick H.;Balbo, Andrea;Schuck, Peter
沉降速度分析超速离心经历了重大转变，这是由于拉姆方程解与实验数据有效拟合的可能性而促成的。这种方法的精度取决于解释实验缺陷的能力，包括样品和仪器。在目前的工作中，我们更详细地探讨了沉降过程、其检测以及数学数据分析中使用的模型之间的关系。我们专注于产生陡峭且快速移动的沉降边界的配置，例如在研究大型多蛋白质复合物时经常遇到的情况。首先，作为一种促进异质样本分析的计算工具，我们引入了部分边界建模策略。它可以通过将直接边界分析限制为沉降系数在预定义范围内的物种来简化建模。接下来，我们研究与实验检测相关的因素，包括高蛋白质浓度下离焦溶液柱产生的光学像差的大小、实验记录的弯月面特征与数据分析中的弯月面参数之间的关系，以及吸光度光学扫描系统的有限径向和时间分辨率的后果。令人惊讶的是，我们发现商用吸收光学器件的有限扫描速度可能会导致较大的误差，超出测量的沉降系数的统计误差一个数量级以上。我们描述了如何在 SEDFIT 和 SEDPHAT 中通过计算来解释这些影响。
Sedimentation velocity analytical ultracentrifugation has experienced a significant transformation, precipitated by the possibility of efficiently fitting Lamm equation solutions to the experimental data. The precision of this approach depends on the ability to account for the imperfections of the experiment, both regarding the sample and the instrument. In the present work, we explore in more detail the relationship between the sedimentation process, its detection, and the model used in the mathematical data analysis. We focus on configurations that produce steep and fast-moving sedimentation boundaries, such as frequently encountered when studying large multi-protein complexes. First, as a computational tool facilitating the analysis of heterogeneous samples, we introduce the strategy of partial boundary modeling. It can simplify the modeling by restricting the direct boundary analysis to species with sedimentation coefficients in a predefined range. Next, we examine factors related to the experimental detection, including the magnitude of optical aberrations generated by out-of-focus solution columns at high protein concentrations, the relationship between the experimentally recorded signature of the meniscus and the meniscus parameter in the data analysis, and the consequences of the limited radial and temporal resolution of the absorbance optical scanning system. Surprisingly, we find that large errors can be caused by the finite scanning speed of the commercial absorbance optics, exceeding the statistical errors in the measured sedimentation coefficients by more than an order of magnitude. We describe how these effects can be computationally accounted for in SEDFIT and SEDPHAT.
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影响因子：
2.9
作者：
GROPPER, L
通讯作者：
GROPPER, L
影响因子：
6.2
作者：
Brown, Patrick H.;Balbo, Andrea;Schuck, Peter
通讯作者：
Schuck, Peter
影响因子：
3.4
作者：
Brown, Patrick H.;Schuck, Peter
通讯作者：
Schuck, Peter
DOI：
10.1208/s12248-008-9058-z
发表时间：
2008-09
期刊：
The AAPS journal
影响因子：
--
作者：
Brown PH;Balbo A;Schuck P
通讯作者：
Schuck P
影响因子：
15
作者：
McCammon, MG;Hernández, H;Robinson, CV
通讯作者：
Robinson, CV