第六章 蛋白质基本性质的分析2016秋

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Titration Curve
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Composition
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Method Outline .pao
• Apply to other proteins • Save different analysis method and its preset patterns (line color, line weight, fill pattern, fill color)
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Coiled-coil
• PROTEAN首先给每个氨基酸赋予相对的出现缠绕卷曲结构的 频率。频率值来自一个做过统计的已知出现缠绕螺旋频率 的数据库。获得相对频率值后，残基被分配到 28个残基的 滑动窗口。这一长度值是呈现稳定的缠绕卷曲结构的四个 和五个七联肽的最小值。将所有的窗口的值相乘，积取 28 为根的对数。每个残基的起始最高分为196。得到的数值以 峰图或区域图表示。
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Charge density
• 电荷平均值法预测正电或负电区域是通过计算在特定范围内的残 基的电荷数的加和来实现的。该方法结果是以一张平均电荷的峰 图和两张电荷分布区域图来表示的，分别是正电区域和负电区域 分布图。由于带电的残基有着位于蛋白表面的趋势，因此，这种 方法有有助于预测蛋白的表面特征。 DNASTAR 软件使用White, Handler and Smith (1964)的pK值表来进行表面特征的预测。同 时，pK值表也被用于测定蛋白和多肽序列的滴定曲线，获得其等 电点（pI）。 • 参数：用于计算电荷平均值的氨基酸残基的数量，即范围窗口的 大小。如果窗口值设得太低，则高带电的残基（如赖氨酸和半胱 氨酸）将产生很大的噪音；如果太高则得到的带电区域太少而只 得到平平的区域图。 • pH: 用于计算每个残基的pK值；正电阈值用于表示正电区域的最 小值，负电阈值则相反。
Method curtain
Analysis surface
Legend curtain
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Two selection
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Add a known feature
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Join a feature
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A Microscope is provided for situations where you want to see the sequence responsible for a method's result. This lets you stay zoomed out for the overall big picture, but still see critical residues in a region. Click the Microscope palette tool to activate its display window. You have your choice of displaying the sequence as chemical formula or space-filling models. Just click the appropriate button in the upper left corner of the Microscope window (as shown in the following figures). The arrow box in the lower left corner controls the size of the amino acid sequence characters and the vertical size of the Microscope window. The arrow box in the lower right corner controls the horizontal size of the Microscope window. Drag either box to control the size of the mini-window. As the cursor's position changes over the assay surface, PROTEAN updates the Microscope window to display residues underneath. You can examine the entire sequence by choosing either Linear Space Fill or Chemical Formula from the Model Structure submenu (under the Analysis menu).
第六章 蛋白质基本性质的分析
2016-11-17
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本章的主要内容
一、蛋白质理化性质的分析 二、酶切图谱
三、亲疏水性分析
四、抗原性分析 五、表面分析 六、柔性分析 七、二级结构预测
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分析界面
• The Assay Document is the file created and used by PROTEAN to examine and elucidate protein sequences. It is often referred to as simply an assay. The assay is composed of five principle parts: the status area, the palette tools, the Method Curtain, the assay surface and the Legend Curtain. Click in the assay graphic below for more on each part.
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Model structure -- Helical Wheel
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Model structure -- Helical Net
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Model structure -- Beta Net
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Model structure – Space Fill Model
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Model structure – Chemical Formula
Using the Microscope
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Tabular Data
• The first column shows an amino acid, the second its position and the remaining columns show the value assigned for each residue at the given position. Double-click any value or column to show the parameter window for the method in question. Changing the order of plots on the assay rearranges the column summaries on the Tabular Data window.
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• Parameters:The only parameter involved in Garnier-Robson predictions is the use of decision constants. If you assign decision constants, you should have prior knowledge of the circular dichroism data. No Decision Constants makes no assumptions of the global a-helix and, βsheet content. Calculated Decision Constants are computer derived from global a-helix and βsheet probabilities. Constants added are based on three protein classes: proteins with less than 20%, between 20% and 50% and over 50% a-helix or β-sheet. Specified Decision Constants are user defined, from circular dichroism(CD) data. • Limitations:This method is a statistical approach, based on observed residue patterns in 25 proteins. If your protein differs substantially from proteins used to establish the model secondary structure frequencies, this method may give inaccurate structural predictions.
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Coiled-coil
• 参数：只有一个确定出现区域图的阈值变量。1.3被 认为是已知的缠绕卷曲的最小值。如果是找象球蛋白 中的不严格的缠绕卷曲结构可以降至1.1；如果是纤 维蛋白的严格缠绕卷曲结构则可升至1.5。 • 限制性：该方法是统计得到的，基于已知的翻译 GenBank和已知的缠绕卷曲结构的数据。如果我们的 蛋白与用于检验的测试值有很大的出入，这方法就不 能准确地预测。再者，虽然我们也可以找到三个或四 个的螺旋并排缠绕，但该方法还是不推荐用于其他类 似非极性七联肽重复区的其他结构。可以结合 Goldman-Engleman-Steitz法预测α螺旋的穿膜区， 也可优化GES方法得到两性（amphipathy）分析。
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二级结构预测(2)： Garnier-Robson
• 该算法通过蛋白的氨基酸序列预测其二级结构。它是基于已知蛋 白的晶体结构的统计方法，尤其是针对二面角 (C‘-N-C-C’-N) 和 氢键网络对α-helical (H), β-pleated sheet (E - extended chains), β-turns (T) 和 coil (C)的区域进行优化。. • Garnier-Robson方法主要考察存在与一定已知结构的特定残基的 倾向性。在赋予最可能的残基构象之前，先考察周围的16个残基 （上8个，下8个），如果倾向于某种特定的结构，初始的残基就 会被归于那种类型的结构，否则，重新评估成其它型的结构。
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Protease map
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Different protease sites MS Degradation PMF (Peptide Map Finger)
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Analysis methods
• • • • • • • • • Protease map Pattern: Prosite database, Ariadne file Charge density Secondary Structure: Coiled Coil, Garnier-Robson, Deleage & Roux, ChouFasman Hydropathy: Goldman-Engleman-Steitz, Kyte-Doolittle, Hopp-Woods Antigenicity: Settle MHC Motifs, AMPHI, Rothbard-Taylor, Jameson-Wolf Amphilicity – Eisenberg Surface Probability – Emini Flexibility – Karplus-Schulz
• 局限性：该方法可能对于参数的改变特别的敏感，因此，使用时 可多尝试改变参数得到多个分布图，以显示细微的差别。
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Charge density
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Charge density
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二级结构预测(1)：Coiled-coil
• 该方法是根据Parry (1982)的方法进行缠绕卷曲结构的预测。 该结构由两个右旋螺旋以堆积角度为20度相互围绕一个左旋超卷 曲形成的。这些螺旋是由侧链基团相互之间的亲水作用稳定着的。 螺旋的特征是非极性氨基酸残基以每圈3.5个的有规律的七联子 周期排布形成。这种规律使得预测缠绕的卷曲结构成为可能。 Myosins（肌球蛋白）和keratins（角蛋白）是这种四级结构元 素的典型。