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Miniproteins: Folding Equilibria, Pathways and Rates

微蛋白：折叠平衡、途径和速率

基本信息

批准号：
7883714
负责人：
Niels Hjorth Andersen
金额：
$ 12.47万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-03 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7883714
关键词：
Address Amino Acids Architecture Behavior Binding Burial Cations Characteristics Chemicals Complex Data Dependence Disease Equilibrium Event Fluorescence Fluorine Fluorocarbons Genetic Crossing Over Glycine Hand Hydrogen Bonding Hydrophobicity Individual Indoles Laboratories Left Length Measures Methods Modeling Molecular Monitor Mutate Mutation Nature Pathway interactions Peptides Physiologic pulse Population Positioning Attribute Process Protein Engineering Proteins Protons RBM5 gene Relative (related person)Reporting Research Personnel Role Running Shapes Side Site Solvents Source Specificity Structure Suggestion Surveys System Techniques Tertiary Protein Structure Vertebral column alpha helix analog base design design and construction fascinate insight melting molecular dynamics mutant novel peptide structure polypeptide preference prolylglycine protein aminoacid sequence protein folding receptor segregation simulation

项目摘要

DESCRIPTION (provided by applicant): The efficient folding of polypeptide sequences into stable structures is one of the most fascinating and fundamentally important biorecognition phenomena. Studies of fold stability as well as folding pathways and rates are often more incisive when performed on minimalist constructs rather than full-sized proteins. The designed Trp-cage fold, consisting of only 18-19 essential residues, has proven to be an excellent system for measuring the fold stabilization contributions of intrinsic secondary structure preferences and individual hydrophobic, coulombic (saltbridge), and H-bonding interactions. Additional mutational studies of the Trp-cage are proposed. These, in conjunction with molecular dynamics simulations of folding trajectories, are aimed at determining, in detail, the pathway(s) of Trp-cage folding and at discovering alternate packing arrangements that could serve as the hydrophobic cores for related miniprotein motifs. Folding rates and cooperativity will be determined by several techniques (NMR line broadening, NMR chemical shift melts, and fluorescence-monitored T-jumps) to ascertain if the rates are dependent on contact order at the lower range of protein size. Extensively-mutated Trp-cage constructs, truncations and circular permutants will figure heavily in these studies. The present proposal continues to address polypeptide structuring requisites by both de novo design and as a fold optimization problem, but with an increasing emphasis on folding pathways and rates. The folding rate determinations should define the source (increased folding rate or decreased unfolding rate) of each of the structure-stabilizing interactions in the Trp-cage. Mutational effects on the rates of b-hairpin and three-stranded sheet formation will also be determined using dynamic NMR methods. Binding and novel structure stabilizing effects of the Trp side chain will be explored and quantitated. The design, construction, and optimization of a BaB3 miniprotein (a parallel b sheet resulting from the association of b strands at each end of an a helix) are also proposed. This construct mimics some features of the Bl domain of protein L. The designed BaB target fold is, however, novel in two major respects: the helix runs in the opposite direction of that in the Bl domain, and there is a left-handed crossover from a B strand to an alpha helix - the latter has never been observed in nature. The studies outlined in this proposal will provide structural and mechanistic insights concerning the requisites for fast and efficient folding that should yield protein engineering strategies for reducing disease-related misfolding events. More accurate methods for quantitating polypeptide structuring and folding rates will also result.

描述（由申请人提供）：多肽序列有效折叠成稳定结构是最吸引人和最重要的生物识别现象之一。折叠稳定性以及折叠途径和速率的研究通常在最低限度的构建体而不是全尺寸蛋白质上进行时更深入。设计的Trp笼折叠，仅由18-19个必需残基组成，已被证明是用于测量固有二级结构偏好和个体疏水性、库仑（盐桥）和H-键合相互作用的折叠稳定贡献的优异系统。建议对Trp笼进行额外的突变研究。这些，结合分子动力学模拟的折叠轨迹，旨在确定，详细，色氨酸笼折叠的途径（S），并在发现替代的包装安排，可以作为相关的miniprotein基序的疏水核心。折叠速率和协同性将通过几种技术（NMR谱线加宽、NMR化学位移熔解和荧光监测的T跳跃）确定，以确定速率是否取决于蛋白质大小较低范围的接触顺序。广泛突变的Trp-笼构建体、截短体和环状置换体将在这些研究中大量出现。本提案继续通过从头设计和作为折叠优化问题来解决多肽结构化问题，但越来越强调折叠途径和速率。折叠速率测定应确定Trp笼中每个结构稳定相互作用的来源（增加的折叠速率或降低的解折叠速率）。还将使用动态NMR方法确定突变对b-发夹和三链片层形成速率的影响。将探索和量化Trp侧链的结合和新型结构稳定作用。还提出了BaB 3微蛋白（a螺旋两端的B链结合形成的平行B层）的设计、构建和优化。该构建体模拟蛋白L的B1结构域的一些特征。然而，设计的Ba B靶折叠在两个主要方面是新颖的：螺旋以与B1结构域中相反的方向运行，并且存在从B链到α螺旋的左手交叉-后者在自然界中从未观察到。本提案中概述的研究将提供有关快速有效折叠的结构和机制的见解，这些见解应产生用于减少疾病相关错误折叠事件的蛋白质工程策略。还将产生用于定量多肽结构化和折叠速率的更准确的方法。

项目成果

期刊论文数量（25）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural insights for designed alanine-rich helices: comparing NMR helicity measures and conformational ensembles from molecular dynamics simulation.

DOI：
10.1002/bip.21004
发表时间：
2008-09
期刊：
BIOPOLYMERS
影响因子：
2.9
作者：
Song, Kun;Stewart, James M.;Fesinmeyer, R. Matthew;Andersen, Niels H.;Simmerling, Carlos
通讯作者：
Simmerling, Carlos

β-Sheet 13C structuring shifts appear only at the H-bonded sites of hairpins.

β-Sheet 13C 结构变化仅出现在发夹的 H 键位点。

DOI：
10.1021/ja1088953
发表时间：
2011
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Shu,Irene;Stewart,JamesM;Scian,Michele;Kier,BrandonL;Andersen,NielsH
通讯作者：
Andersen,NielsH

Optimization of a β-sheet-cap for long loop closure.

DOI：
10.1002/bip.22995
发表时间：
2017-03
期刊：
Biopolymers
影响因子：
2.9
作者：
Anderson JM;Shcherbakov AA;Kier BL;Kellock J;Shu I;Byrne AL;Eidenschink LA;Andersen NH
通讯作者：
Andersen NH

Quantitating amino acid beta-strand preferences, turn propensities and cross-strand interactions in a designed hairpin peptide.

定量设计的发夹肽中的氨基酸 β 链偏好、转向倾向和跨链相互作用。