Learning To Fold Proteins Using Energy Landscape Theory

This review is a tutorial for scientists interested in the problem of protein structure prediction, particularly those interested in using coarse-grained molecular dynamics models which are optimized using lessons learned from the energy landscape theory of protein folding. We also present a review of the results of the AMH/AMC/AMW/AWSEM family of coarse-grained molecular dynamics protein folding models to illustrate the points covered in the first part of the article. Accurate coarse-grained structure prediction models can be used to investigate a wide range of conceptual and mechanistic issues outside of protein structure prediction; specifically, the paper concludes by reviewing how AWSEM has, in recent years, been able to elucidate questions related to the unusual kinetic behavior of artificially designed proteins, multidomain protein misfolding, and the initial stages of protein aggregation.     

A Statistical Analysis of the Sequence and Structure of Thermophilic and Non-Thermophilic Proteins

Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial scale has been increasing; therefore, the engineering of thermophilic proteins has become a hot direction in the field of protein engineering. However, the exact mechanism of thermostability of proteins is not yet known, for engineering thermophilic proteins knowing the basis of thermostability is necessary. In order to understand the basis of the thermostability in proteins, we have made a statistical analysis of the sequences, secondary structures, hydrogen bonds, salt bridges, DHA (Donor–Hydrogen–Accepter) angles, and bond lengths of ten pairs of thermophilic proteins and their non-thermophilic orthologous. Our findings suggest that polar amino acids contribute to thermostability in proteins by forming hydrogen bonds and salt bridges which provide resistance against protein denaturation. Short bond length and a wider DHA angle provide greater bond stability in thermophilic proteins. Moreover, the increased frequency of aromatic amino acids in thermophilic proteins contributes to thermal stability by forming more aromatic interactions. Additionally, the coil, helix, and loop in the secondary structure also contribute to thermostability.     

Going with the Electron Flow: Heme Electronic Structure and Electron Transfer in Cytochrome c

Heme is an essential and functionally versatile cofactor. Our understanding of how the environment of a heme in a protein tunes its function has benefited from spectroscopic and functional investigations of heme proteins and their variants with altered heme environments. Two properties of current interest are the conformation of the heme and hydrogen bonding to heme propionates. By combining nuclear magnetic resonance experiments and density functional theory calculations, both of these characteristics have been shown to influence the distribution of the singly occupied molecular orbital on the heme of ferricytochrome c, which affects coupling to redox partners and electron-transfer rates. In addition, heme conformation has been shown to tune reduction potential. These results reveal that subtle variations in heme conformation and in interactions with its propionates can have significant impacts on electron-transfer activity.     

Distance-Dependent Electronic Interactions Across DNA Base Pairs: Charge Transport,Exciton Coupling,and Energy Transfer

The π-stacked bases of B-DNA constitute a helical ruler present nowhere else in nature. When two chromophores are placed at the ends of a base-pair domain with fixed orientations of their transition dipole moments, insertion of an additional base pair between the chromophores increases the distance between the chromophores by ca. 3.4 Å and the angle between their transition dipoles by ca. 36°. The consequences of these changes on the dynamics and efficiency of photoinduced charge transport and upon exciton-coupled circular dichroism and fluorescence resonance energy transfer are discussed.     

2-脲基-4[1氢]-嘧啶酮四氢键萘-蒽超分子组装体系:“通过键”机制的单重态能量传递

设计合成了基于2-脲基-4[1氢]-嘧啶酮AADD四氢键萘-蒽超分子组装体系UPNa.UPAn.稳态和时间分辨荧光光谱研究表明UPNa.UPAn四氢键组装体可以发生从萘到蒽的高效、快速的单重态能量传递过程.体系内光诱导单重态能量传递的速度和效率远大于通过F rster机制的单重态能量传递速率,表明组装体系UPNa.UPAn中萘向蒽的光诱导单重态能量传递是经由体系内的F rster和通过AADD四氢键的超交换机制发生的.     

Exploring Nearest Neighbor Interactions and Their Influence on the Gibbs Energy Landscape of Unfolded Proteins and Peptides

The Flory isolated pair hypothesis (IPH) is one of the corner stones of the random coil model, which is generally invoked to describe the conformational dynamics of unfolded and intrinsically disordered proteins (IDPs). It stipulates, that individual residues sample the entire sterically allowed space of the Ramachandran plot without exhibiting any correlations with the conformational dynamics of its neighbors. However, multiple lines of computational, bioinformatic and experimental evidence suggest that nearest neighbors have a significant influence on the conformational sampling of amino acid residues. This implies that the conformational entropy of unfolded polypeptides and proteins is much less than one would expect based on the Ramachandran plots of individual residues. A further implication is that the Gibbs energies of residues in unfolded proteins or polypeptides are not additive. This review provides an overview of what is currently known and what has yet to be explored regarding nearest neighbor interactions in unfolded proteins.     

Photoswitchable Fluorescent Proteins: Mechanisms on Ultrafast Timescales

The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore cis-trans isomerization and proton transfer for negative and positive RSFPs and hydration–dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing various ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time “action” details regarding the bidirectional cis-trans isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors.     

Exploring Large Domain Motions in Proteins Using Atomistic Molecular Dynamics with Enhanced Conformational Sampling

Conformational transitions in multidomain proteins are essential for biological functions. The Apo conformations are typically open and flexible, while the Holo states form more compact conformations stabilized by protein-ligand interactions. Unfortunately, the atomically detailed mechanisms for such open-closed conformational changes are difficult to be accessed experimentally as well as computationally. To simulate the transitions using atomistic molecular dynamics (MD) simulations, efficient conformational sampling algorithms are required. In this work, we propose a new approach based on generalized replica-exchange with solute tempering (gREST) for exploring the open-closed conformational changes in multidomain proteins. Wherein, selected surface charged residues in a target protein are defined as the solute region in gREST simulation and the solute temperatures are different in replicas and exchanged between them to enhance the domain motions. This approach is called gREST selected surface charged residues (gREST_SSCR) and is applied to the Apo and Holo states of ribose binding protein (RBP) in solution. The conformational spaces sampled with gREST_SSCR are much wider than those with the conventional MD, sampling open-closed conformational changes while maintaining RBP domains’ stability. The free-energy landscapes of RBP in the Apo and Holo states are drawn along with twist and hinge angles of the two moving domains. The inter-domain salt-bridges that are not observed in the experimental structures are also important in the intermediate states during the conformational changes.     

Hidden Relationships between N-Glycosylation and Disulfide Bonds in Individual Proteins

N-Glycosylation (NG) and disulfide bonds (DBs) are two prevalent co/post-translational modifications (PTMs) that are often conserved and coexist in membrane and secreted proteins involved in a large number of diseases. Both in the past and in recent times, the enzymes and chaperones regulating these PTMs have been constantly discovered to directly interact with each other or colocalize in the ER. However, beyond a few model proteins, how such cooperation affects N-glycan modification and disulfide bonding at selective sites in individual proteins is largely unknown. Here, we reviewed the literature to discover the current status in understanding the relationships between NG and DBs in individual proteins. Our results showed that more than 2700 human proteins carry both PTMs, and fewer than 2% of them have been investigated in the associations between NG and DBs. We summarized both these proteins with the reported relationships in the two PTMs and the tools used to discover the relationships. We hope that, by exposing this largely understudied field, more investigations can be encouraged to unveil the hidden relationships of NG and DBs in the majority of membranes and secreted proteins for pathophysiological understanding and biotherapeutic development.     

Influences of Electromagnetic Energy on Bio-Energy Transport through Protein Molecules in Living Systems and Its Experimental Evidence

The influences of electromagnetic fields (EMFs) on bio-energy transport and its mechanism of changes are investigated through analytic and numerical simulation and experimentation. Bio-energy transport along protein molecules is performed by soliton movement caused by the dipole–dipole electric interactions between neighboring amino acid residues. As such, EMFs can affect the structure of protein molecules and change the properties of the bio-energy transported in living systems. This mechanism of biological effect from EMFs involves the amino acid residues in protein molecules. To study and reveal this mechanism, we simulated numerically the features of the movement of solitons along protein molecules with both a single chain and with three channels by using the Runge–Kutta method and Pang’s soliton model under the action of EMFs with the strengths of 25,500, 51,000, 76,500, and 102,000 V/m in the single-chain protein, as well as 17,000, 25,500, and 34,000 V/m in the three-chain protein, respectively. Results indicate that electric fields (EFs) depress the binding energy of the soliton, decrease its amplitude, and change its wave form. Also, the soliton disperses at 102,000 V/m in a single-chain protein and at 25,500 and 34,000 V/m in three-chain proteins. These findings signify that the influence of EMFs on the bio-energy transport cannot be neglected; however, these variations depend on both the strength and the direction of the EF in the EMF. This direction influences the biological effects of EMF, which decrease with increases in the angle between the direction of the EF and that of the dipole moment of amino acid residues; however, randomness at the macroscopic level remains. Lastly, we experimentally confirm the existence of a soliton and the validity of our conclusion by using the infrared spectra of absorption of the collagens, which is activated by another type of EF. Thus, we can affirm that both the described mechanism and the corresponding theory are correct and that EMFs or EFs can influence the features of energy transport in living systems and thus have certain biological effects.     

Simulating Biomolecular Folding and Function by Native-Structure-Based/Go-Type Models

The 2013 Nobel Prize in Chemistry highlights how crucial computer simulations have become for many scientific and engineering fields. Nowadays, scientific progress is not only driven by the interplay of new experimental measurements and increasingly sophisticated theoretical frameworks, but also by an incredible toolbox of complex computational models meeting ubiquitously available computing power and data storage facilities. Quantum mechanical (QM) calculations can be condensed into molecular mechanics (MM) force fields and coupled QM/MM calculations can derive atomic and molecular properties of biomolecular or materials science systems with high accuracy. Pure MM simulations driven by Monte Carlo or molecular dynamics algorithms are widely applied in biological chemistry/physics and can investigate large biomolecular systems, such as proteins, DNA, or RNA. One coarse-grained class of these models, native-structure-based or Go models, are based on energy landscape theory and the principle of minimal frustration. Herein, an ensemble of converging pathways guide protein folding on a funnel-like shape of the entire energy landscape towards the native state. Simulations based on these ideas have been tremendously successful in explaining protein folding and function. Their history and recent application highlights are reviewed.     

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

Tetraphenylporphyrin Enters the Ring: First Example of a Complex between Highly Bulky Porphyrins and a Protein**

Tetraphenylporphyrin (TPP) is a symmetrically substituted synthetic porphyrin whose properties can be readily modified, providing it with significant advantages over naturally occurring porphyrins. Herein, we report the first example of a stable complex between a native biomolecule, the haemoprotein HasA, and TPP as well as its derivatives. The X-ray crystal structures of nine different HasA-TPP complexes were solved at high resolutions. HasA capturing TPP derivatives was also demonstrated to inhibit growth of the opportunistic pathogen Pseudomonas aeruginosa. Mutant variants of HasA binding FeTPP were shown to possess a different mode of coordination, permitting the cyclopropanation of styrene.     

Structure and Thermal Stability of wtRop and RM6 Proteins through All-Atom Molecular Dynamics Simulations and Experiments

In the current work we study, via molecular simulations and experiments, the folding and stability of proteins from the tertiary motif of 4-α-helical bundles, a recurrent motif consisting of four amphipathic α-helices packed in a parallel or antiparallel fashion. The focus is on the role of the loop region in the structure and the properties of the wild-type Rop (wtRop) and RM6 proteins, exploring the key factors which can affect them, through all-atom molecular dynamics (MD) simulations and supporting by experimental findings. A detailed investigation of structural and conformational properties of wtRop and its RM6 loopless mutation is presented, which display different physical characteristics even in their native states. Then, the thermal stability of both proteins is explored showing RM6 as more thermostable than wtRop through all studied measures. Deviations from native structures are detected mostly in tails and loop regions and most flexible residues are indicated. Decrease of hydrogen bonds with the increase of temperature is observed, as well as reduction of hydrophobic contacts in both proteins. Experimental data from circular dichroism spectroscopy (CD), are also presented, highlighting the effect of temperature on the structural integrity of wtRop and RM6. The central goal of this study is to explore on the atomic level how a protein mutation can cause major changes in its physical properties, like its structural stability.     

Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring,Signal Communication and Body Energy Sensing

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.