使用胺N CEST NMR实验,研究"可见"蛋白质状态之间的缓慢交换
Nihar Pradeep Khandave1, Ved Prakash Tiwari1, Pramodh Vallurupalli1
1Tata Institute of Fundamental Research Hyderabad, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad 500046, India.
概括
这项研究介绍了15N CEST作为分析蛋白质动态的ZZ交换NMR的优质替代品. 15N CEST实验有效地克服了光谱重叠,使得蛋白质状态交换的精确表征成为可能.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 核磁共振 (NMR) 光谱学 核磁共振 (NMR) 光谱学
背景情况:
- 蛋白质状态之间的缓慢交换对于理解蛋白质功能和动态至关重要.
- 传统的2D ZZ交换NMR实验可能会出现光谱重叠,使分析复杂化.
- 需要使用替代方法来准确研究蛋白质动力学和结构变化.
研究的目的:
- 评估15N CEST实验作为1H-15N ZZ交换NMR的替代方案,用于研究蛋白质状态之间的缓慢交换.
- 为了证明15N CEST如何克服ZZ交换实验中固有的光谱重叠问题.
- 建立一种使用15N CEST. 获取特定站点交换参数的方法.
主要方法:
- 利用15N CEST实验研究蛋白质状态之间的交换.
- 在双状态交换系统中记录了每个交换站点的两个15N CEST配置文件.
- 同时分析了15N CEST配置文件与初始磁化或小状态人口数据.
- 将该方法应用于T4溶酶的T34A突变,该突变在原生和次要状态之间进行相互转换.
主要成果:
- 15N CEST实验有效地避免了ZZ交换NMR中遇到的光谱重叠.
- 通过分析双重15N CEST配置文件,准确确定特定站点的交换参数.
- 该方法成功地描述了T4 lysoszyme突变体 (18kDa) 在其原生状态和小状态之间 (21%的人口,在40°C时~5s-1的交换率) 的交换动态.
结论:
- 15N CEST是ZZ交换NMR的一个强大而有利的替代方案,用于研究缓慢的蛋白质动力学.
- 这种方法提供了一种可靠的方法,以高精度描述"可见"蛋白质状态之间的交换.
- 这项研究验证了15N CEST的实用性,用于详细分析蛋白质构造交换过程.
相关概念视频
01:29Preparation of Amides
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Amides are synthesized by treating carboxylic acids with amines in the presence of dehydrating agents like dicyclohexylcarbodiimide (DCC).
The DCC-promoted synthesis of amides begins with the protonation of DCC by carboxylic acid. The protonation makes it a better acceptor. Next, the addition of carboxylate to the protonated carbodiimide gives a reactive acylating agent.
Subsequently, the amine acts as a nucleophile that attacks the acylating agent to form a tetrahedral intermediate. In the...
The DCC-promoted synthesis of amides begins with the protonation of DCC by carboxylic acid. The protonation makes it a better acceptor. Next, the addition of carboxylate to the protonated carbodiimide gives a reactive acylating agent.
Subsequently, the amine acts as a nucleophile that attacks the acylating agent to form a tetrahedral intermediate. In the...
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01:19Amines to Amides: Acylation of Amines
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Various carboxylic acid derivatives (such as acid chlorides, esters, and anhydrides) can be used for the acylation of amines to yield amides. The reaction requires two equivalents of amines. The first amine molecule functions as a nucleophile and attacks the carbonyl carbon to produce a tetrahedral intermediate. This is followed by the loss of the leaving group and restoration of the C=O bond.
Next, the second equivalent of amine serves as a Brønsted base and deprotonates the quaternary...
Next, the second equivalent of amine serves as a Brønsted base and deprotonates the quaternary...
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01:28Amides to Carboxylic Acids: Hydrolysis
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Amides can undergo either acid-catalyzed hydrolysis or base-promoted hydrolysis through a typical nucleophilic acyl substitution. Each hydrolysis requires severe conditions.
Acid-catalyzed hydrolysis:
Hydrolysis of amides under acidic conditions yields carboxylic acids. Since the reaction occurs slowly, hydrolysis requires the conditions of heat.
The mechanism begins with the protonation of the carbonyl oxygen by the acid catalyst. The protonation makes the amide carbonyl carbon more...
Acid-catalyzed hydrolysis:
Hydrolysis of amides under acidic conditions yields carboxylic acids. Since the reaction occurs slowly, hydrolysis requires the conditions of heat.
The mechanism begins with the protonation of the carbonyl oxygen by the acid catalyst. The protonation makes the amide carbonyl carbon more...
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01:07Acid Halides to Amides: Aminolysis
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Aminolysis is a nucleophilic acyl substitution reaction, where ammonia or amines act as nucleophiles to give the substitution product. Acid halides react with ammonia, primary amines, and secondary amines to yield primary, secondary, and tertiary amides, respectively.
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
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01:22Preparation of 1° Amines: Azide Synthesis
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Direct alkylation of ammonia produces polyalkylated amines, along with a quaternary ammonium salt. To exclusively prepare primary amines, the azide synthesis method can be used.
Azide ions act as good nucleophiles and react with unhindered alkyl halides to form alkyl azides. Alkyl azides do not participate in further nucleophilic substitution reactions, thereby eliminating the chances of polyalkylated products. Alkyl azides are reduced by hydride-based reducing agents, like lithium aluminum...
Azide ions act as good nucleophiles and react with unhindered alkyl halides to form alkyl azides. Alkyl azides do not participate in further nucleophilic substitution reactions, thereby eliminating the chances of polyalkylated products. Alkyl azides are reduced by hydride-based reducing agents, like lithium aluminum...
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01:11Nomenclature of Carboxylic Acid Derivatives: Amides and Nitriles
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Naming Amides
The IUPAC and common names of amides are derived from the parent carboxylic acid, by replacing the suffix “oic acid” and “ic acid,” respectively, with “amide.” In the following example, the IUPAC name ethanamide is derived from ethanoic acid, and the common name, acetamide, is obtained from acetic acid.
The IUPAC and common names of amides are derived from the parent carboxylic acid, by replacing the suffix “oic acid” and “ic acid,” respectively, with “amide.” In the following example, the IUPAC name ethanamide is derived from ethanoic acid, and the common name, acetamide, is obtained from acetic acid.
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