Structural Disorder of Folded Proteins: Isotope-Edited 2D IR Spectroscopy and Markov State Modeling

The conformational heterogeneity of the N-terminal domain of the ribosomal protein L9 (NTL9_1-39) in its folded state is investigated using isotope-edited two-dimensional infrared spectroscopy. Backbone carbonyls are isotope-labeled (¹³C=¹⁸O) at five selected positions (V3, V9, V9G13, G16, and G24) to provide a set of localized spectroscopic probes of the structure and solvent exposure at these positions. Structural interpretation of the amide I line shapes is enabled by spectral simulations carried out on structures extracted from a recent Markov state model. The V3 label spectrum indicates that the β-sheet contacts between strands I and II are well folded with minimal disorder. The V9 and V9G13 label spectra, which directly probe the hydrogen-bond contacts across the β-turn, show significant disorder, indicating that molecular dynamics simulations tend to overstabilize ideally folded β-turn structures in NTL9_1-39. In addition, G24-label spectra provide evidence for a partially disordered α-helix backbone that participates in hydrogen bonding with the surrounding water.     

Visualization of Protein Folding Funnels in Lattice Models

Protein folding occurs in a very high dimensional phase space with an exponentially large number of states, and according to the energy landscape theory it exhibits a topology resembling a funnel. In this statistical approach, the folding mechanism is unveiled by describing the local minima in an effective one-dimensional representation. Other approaches based on potential energy landscapes address the hierarchical structure of local energy minima through disconnectivity graphs. In this paper, we introduce a metric to describe the distance between any two conformations, which also allows us to go beyond the one-dimensional representation and visualize the folding funnel in 2D and 3D. In this way it is possible to assess the folding process in detail, e.g., by identifying the connectivity between conformations and establishing the paths to reach the native state, in addition to regions where trapping may occur. Unlike the disconnectivity maps method, which is based on the kinetic connections between states, our methodology is based on structural similarities inferred from the new metric. The method was developed in a 27-mer protein lattice model, folded into a 3×3×3 cube. Five sequences were studied and distinct funnels were generated in an analysis restricted to conformations from the transition-state to the native configuration. Consistent with the expected results from the energy landscape theory, folding routes can be visualized to probe different regions of the phase space, as well as determine the difficulty in folding of the distinct sequences. Changes in the landscape due to mutations were visualized, with the comparison between wild and mutated local minima in a single map, which serves to identify different trapping regions. The extension of this approach to more realistic models and its use in combination with other approaches are discussed.     

Molecular Dynamic Simulation of Chaperonin-Mediated Protein Folding

We use molecular dynamics methods to simulate chaperonin-mediated refolding of barnase. A chaperonin term is added to the force field in order to simulate the hydrophobic environment in the central cavity of the chaperonins. Two aspects of our simulation results are consistent with experiments: (1) The hydrophobic environment of the central cavity of the chaperonin is an advantageous condition for the refolding of the misfolded intermediates. (2) One cycle of binding and release is not enough for the successful folding. Chaperonin-assisted protein folding maybe a procedure of multiple cycles of binding and release from the chaperonin.     

Molecular Dynamics Simulation of Protein Folding with Supersecondary Structure Constraints

We integrate molecular dynamics simulation methods with a newly developed supersecondary structure prediction method and compute the structure of a protein molecule, crambin. The computed structure is similar to the crystal structure with an rms error of 3.94 Å.     

Assembly and Kinetic Folding Pathways of a Tetrameric β-Sheet Complex: Molecular Dynamics Simulations on Simplified Off-Lattice Protein Models

We have performed discontinuous molecular dynamic simulations of the assembly and folding kinetics of a tetrameric β-sheet complex that contains four identical four-stranded antiparallel β-sheet peptides. The potential used in the simulation is a hybrid Go-type potential characterized by the bias gap parameter g, an artificial measure of a model protein's preference for its native state, and the intermolecular contact parameter η, which measures the ratio of intermolecular to intramolecular native attractions. The formation of the β-sheet complex and its equilibrium properties strongly depend on the size of the intermolecular contact parameter η. The ordered β-sheet complex in the folded state and nonaligned β-sheets or tangled chains in the misfolded state are distinguished by measuring the squared radius of gyration and the fraction of native contacts Q. The folding yield for the folded state is high at intermediate values of η, but is low at both small and large values of η. The folded state at small η is liquid-like, but is solid-like at both intermediate and large η. The misfolded state at small η contains nonaligned β-sheets and tangled chains with poor secondary structure at large η. Various folding pathways via dimeric and trimeric intermediates are observed, depending on η. Comparison with experimental results on protein aggregation indicates that intermediate η values are most appropriate for modeling fibril formation and small η values are most appropriate for modeling the formation of amorphous aggregates.     

Calcium-mediated Protein Folding and Stabilization of Salmonella Biofilm-associated Protein A

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~ 386-kDa surface protein, comprising 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium binding and the folded domains have differential mechanical stabilities. Importantly, we identify that > 100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (μM) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion.     

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

In recent years, a growing number of protein folding studies have focused on the unfolded state, which is now recognized as playing a major role in the folding process. Some of these studies show that interactions occurring in the unfolded state can significantly affect the stability and kinetics of the protein folding reaction. In this study, we modeled the effect of electrostatic interactions, both native and nonnative, on the folding of three protein systems that underwent selective charge neutralization or reversal or complete charge suppression. In the case of the N-terminal L9 protein domain, our results directly attribute the increase in thermodynamic stability to destabilization of the unfolded ensemble, reaffirming the experimental observations. These results provide a deeper structural insight into the ensemble of the unfolded state and predict a new mutation site for increased protein stability. In the second case, charge reversal mutations of RNase Sa affected protein stability, with the destabilizing mutations being less destabilizing at higher salt concentrations, indicating the formation of charge-charge interactions in the unfolded state. In the N-terminal L9 and RNase Sa systems, changes in electrostatic interactions in the unfolded state that cause an increase in free energy had an overall compaction effect that suggests a decrease in entropy. In the third case, in which we compared the β-lactalbumin and hen egg-white lysozyme protein homologues, we successfully eliminated differences between the folding kinetics of the two systems by suppressing electrostatic interactions, supporting previously reported findings. Our coarse-grained molecular dynamics study not only reproduces experimentally reported findings but also provides a detailed molecular understanding of the elusive unfolded-state ensemble and how charge-charge interactions can modulate the biophysical characteristics of folding.     

Dynameomics: A Consensus View of the Protein Unfolding/Folding Transition State Ensemble across a Diverse Set of Protein Folds

The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-α, all-β, and mixed α/β and α + β) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an α-helix maintained more structure in the TS ensemble than did residues starting in β-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS.     

模拟蛋白质折叠过程的新算法研究

蛋白质折叠过程模拟是当前蛋白质研究领域的一个难点问题。针对这一问题,提出了一个描述蛋白质折叠过程的算法-拟蛇算法,并且从分子振荡和分子动力学理论两个方面来证明该算法的核心函数是可行和正确的。经过实验总结出所有蛋白质空间结构都可以通过两种类型函数构造出来,提出了描述蛋白质折叠过程模型。与其它蛋白质折叠过程模拟算法的实验结果比较表明,拟蛇算法所构造的空间结构能量值最小、相似度最好。进而说明拟蛇算法和蛋白质折叠过程模型在描述蛋白质折叠过程方面具有明显优势。     

Cystic Fibrosis: A Disease of Altered Protein Folding

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, CFTR. Previously we demonstrated that the common F508 mutation in the first nucleotide binding domain (NBD1) alters the ability of the domain to fold into a functional three-dimensional structure, providing a molecular explanation for the observation that the mutant CFTR is retained in the endoplasmic reticulum and does not traffic to the apical membrane of affected epithelial cells. Notably, when conditions are altered to promote folding of the mutant protein, it can assume a functional conformation. Correcting the folding defect may have therapeutic benefit for the treatment of cystic fibrosis. Here we summarize these results and discuss the implications in vitro folding studies have for understanding the pathobiology of CF.     

A Molecular Clock Regulates Angiopoietin-Like Protein 2 Expression

Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.     

Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.     

How Co-translational Folding of Multi-domain Protein Is Affected by Elongation Schedule: Molecular Simulations

Co-translational folding (CTF) facilitates correct folding in vivo, but its precise mechanism remains elusive. For the CTF of a three-domain protein SufI, it was reported that the translational attenuation is obligatory to acquire the functional state. Here, to gain structural insights on the underlying mechanisms, we performed comparative molecular simulations of SufI that mimic CTF as well as refolding schemes. A CTF scheme that relied on a codon-based prediction of translational rates exhibited folding probability markedly higher than that by the refolding scheme. When the CTF schedule is speeded up, the success rate dropped. These agree with experiments. Structural investigation clarified that misfolding of the middle domain was much more frequent in the refolding scheme than that in the codon-based CTF scheme. The middle domain is less stable and can fold via interactions with the folded N-terminal domain. Folding pathway networks showed the codon-based CTF gives narrower pathways to the native state than the refolding scheme.     

Exploiting Models of Molecular Evolution to Efficiently Direct Protein Engineering

Directed evolution and protein engineering approaches used to generate novel or enhanced biomolecular function often use the evolutionary sequence diversity of protein homologs to rationally guide library design. To fully capture this sequence diversity, however, libraries containing millions of variants are often necessary. Screening libraries of this size is often undesirable due to inaccuracies of high-throughput assays, costs, and time constraints. The ability to effectively cull sequence diversity while still generating the functional diversity within a library thus holds considerable value. This is particularly relevant when high-throughput assays are not amenable to select/screen for certain biomolecular properties. Here, we summarize our recent attempts to develop an evolution-guided approach, Reconstructing Evolutionary Adaptive Paths (REAP), for directed evolution and protein engineering that exploits phylogenetic and sequence analyses to identify amino acid substitutions that are likely to alter or enhance function of a protein. To demonstrate the utility of this technique, we highlight our previous work with DNA polymerases in which a REAP-designed small library was used to identify a DNA polymerase capable of accepting non-standard nucleosides. We anticipate that the REAP approach will be used in the future to facilitate the engineering of biopolymers with expanded functions and will thus have a significant impact on the developing field of ‘evolutionary synthetic biology’.     

15N标记蛋白GAL4(62)的2D NMR实验

利用自编的脉冲程序,采用预饱和和自旋锁定对水峰进行双重抑制的方法,得到了 ¹⁵N标记蛋白GAL4(62)的2D ¹H-¹⁵N HSQC、HSQC-NOESY、HSQC-TOCSY谱,并对这几个谱在蛋白质 ¹H谱的归属中所起的作用进行了讨论.     

^15N标记蛋白GAL4（62）的2D NMR实验

利用自编的脉冲程序,采用预饱和和自旋锁定对水峰进行双重抑制的方法,得到了＾１５Ｎ标记蛋白ＧＡＬ４（６２）的２Ｄ＾１Ｈ－＾１５ＮＨＳＱＣ、ＨＳＱＣ－ＮＯＥＳＹ、ＨＳＱＣ－ＴＯＣＳＹ谱,并对这几个谱在蛋白质＾１Ｈ谱的归属中所到的作用作了讨论。