HYBRID PROTEIN ENGINEERING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 设计一个折叠成所需三维形状的蛋白质序列被称为蛋白质折叠逆问题。在自然界中，蛋白质序列仅限于天然产生的20种氨基酸及其翻译后修饰的组合。非天然氨基酸的加入为设计采用稳定折叠的蛋白质提供了独特的可能性。例如，将氨基异丁酸(AIB)掺入蛋白质中会限制该残基的phi和psi扭转角，从而促进螺旋的形成。人们认为，AIB通过前组织降低了螺旋形成对蛋白质折叠的熵惩罚。根据同样的原理，将a-螺旋、b-折叠和反向旋转的半刚性模拟引入蛋白质中，将通过预组织将折叠过程中的熵损失降至最低，同时保留相互作用的表面特征，这些特征优化了折叠状态下的有利焓相互作用。半刚性模拟物缩小了蛋白质的折叠空间，应该通过成核来促进蛋白质的折叠。模块化的二级结构模拟可以作为构建超稳定的、具有催化活性的抗降解蛋白质的基础。BBA折叠：Dahiyat和Mayo的28个残基FSD-1βBBA蛋白被计算设计成独立于锌结合的稳定的锌指BBA折叠。FSD-1的12个残基的α-螺旋区域将被设计的螺旋模拟物取代，形成一种部分是天然多肽，部分是模拟多肽的杂交蛋白。这种杂交蛋白预计比FSD-1更稳定，因为折叠时的熵惩罚减少了。使用GROMACS3.3在显性水中的分子动力学模拟可以在很长(50-100纳秒)的时间尺度上进行，以预测其稳定性。为了进一步稳定BBA折叠，设计了一种含有连接螺旋多肽的杂合半合成蛋白。核糖核酸酶A-核糖核酸酶是一种能被枯草杆菌酶切成S肽(残基1-20)和S蛋白(残基21-124)的酶。S-肽和S-蛋白本身都不具有酶活性。将缩短的S肽(残基1-14)以1：1的摩尔比重新引入S-蛋白质的溶液中，可以恢复全部的酶活性。这有效地将蛋白质折叠事件(S-肽/S-蛋白质重新结合)映射到宏观读数(酶活性的恢复率)。核糖核酸酶可能是20世纪最彻底的表征酶，它为现有的热力学、构象和动力学结果提供了令人难以置信的基础，可以与新的实验数据进行比较，并能够快速、有效地转换到新的系统。设计了一种新型的以模拟14个氨基酸残基的S多肽为目标的螺旋状模拟多肽分子，以再生酶活性。这一奖项将使人们能够对小分子蛋白质在显性溶剂中的稳定性进行计算机评估，以便设计的杂交蛋白质可以与它们的对照亲本进行比较，从而可以在昂贵的几轮合成和实验测量之前反复评估增强的热稳定性。在这项工作中，我们将在多个温度下对BBA和RNaseA蛋白在显性溶剂中进行50-100 ns的模拟，以估计热变性的温度。拟调和近似将被用来估计构象空间。在奔腾4系统上，模拟1纳秒的溶剂化BBA系统需要12个CPU小时。我们计划用10,000个CPU小时来模拟包括野生型和杂交蛋白在内的BBA系统，并用20,000个CPU小时来模拟RNaseA系统。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Designing a protein sequence which folds into a desired three-dimensional shape is known as the inverse protein-folding problem. In nature, protein sequences are limited to combinations of the naturally occurring 20 amino acids and their post-translational modifications. Incorporation of non-natural amino acids provides unique possibilities for designing proteins that adopt a stable fold. For example, incorporation of Aminoisobutyric acid (Aib) into proteins restricts the phi and psi torsion angles of that residue and is known to promote helix formation. It is believed that Aib lowers the entropic penalty of helix formation upon protein folding through preorganization. By the same principle, incorporating semi-rigid mimetics of a-helices, b-sheets, and reverse-turns into a protein would minimize the entropy loss on folding through preorganization while retaining the interactive surface features that optimize the favorable enthalpic interactions in the folded state. A semi-rigid mimetic reduces a proteins fold space and should promote protein folding by nucleation. Modular secondary structure mimetics can serve as building blocks in the design of ultra-stable, catalytically active proteins that resist degradation. BBA foldamers: The 28-residue FSD-1 betabeatalpha (BBA) protein of Dahiyat and Mayo was computationally designed to form a stable zinc-finger BBA fold independent of zinc binding. The 12 residue alpha-helical region of FSD-1 will be replaced with a designed helix mimetic to form a hybrid protein that is part natural peptide and part peptidomimetic. This hybrid protein is expected to be more stable than FSD-1 because the entropic penalty upon folding is reduced. Molecular dynamics simulations in explicit water using GROMACS 3.3 can be performed on a long (50-100 nanoseconds) time scale to predict its stability. A hybrid semi-synthetic protein containing a ligated helical peptidomimetic aimed at further stabilizing the BBA fold has been designed. Ribonuclease A - RNAse is an enzyme which can be cleaved into S-peptide (residues 1-20) and S-protein (residues 21-124) by subtilisin. Neither S-peptide nor S-protein is enzymatically active on its own. Re-introducing a shortened S-peptide (residues 1-14) into a solution with S-protein at 1:1 molar ratio restores full enzymatic activitythis effectively maps a protein-folding event (S-peptide/S-protein re-association) to a macroscopic readout (percent restoration of enzymatic activity). RNAse is probably the most thoroughly characterized enzyme of the 20th century, providing an incredible foundation of existing thermodynamic, conformational, and kinetic results against which to compare new experimental data and enable quick, valid translation to new systems. A novel helical peptidomimetic molecule aimed at mimicking the 14 residue S-peptide and regenerating enzymatic activity has been designed. This award will enable in silico evaluation of the stability of a small protein in explicit solvent so that designed hybrid proteins can be compared with their control parents so that enhanced thermal stability can be iteratively evaluated prior to expensive rounds of synthesis and experimental measurement. For this work, we will perform 50-100 ns simulations of the BBA and RNAse A proteins in explicit solvent at multiple temperatures to estimate the temperature for thermal denaturation. Quasi-harmonic approximations will be applied to estimate the conformational space. On a Pentium 4 system, it takes 12 CPU hours to simulate one nanosecond of a solvated BBA system. We plan to use 10,000 CPU hours to simulate the BBA systems which includes both wild type and hybrid proteins, and 20,000 CPU hours to simulate the RNASe A systems.