Native Topology Research Articles

Topological principles of protein folding

What is the topology of a protein and what governs protein folding to a specific topology? This is a fundamental question in biology. The protein folding reaction is a critically important cellular process, which is failing in many prevalent diseases. Understanding protein folding is also key to the design of new proteins for applications. However, our ability to predict the folding of a protein chain is quite limited andmuch is still unknown about the topological principles of folding. Current predictors of folding kinetics, including the contact order and size, present a limited predictive power, suggesting that these models are fundamentally incomplete. Here, we use a newly developed mathematical framework to define and extract the topology of a native protein conformation beyond knot theory, and investigate the relationship between native topology and folding kinetics in experimentally characterized proteins. We show that not only the folding rate, but also the mechanistic insight into folding mechanisms can be inferred from topological parameters. We identify basic topological features that speed up or slow down the folding process. The approach enabled the decomposition of protein 3D conformation into topologically independent elementary folding units, called circuits. The number of circuits correlates significantly with the folding rate, offering not only an efficient kinetic predictor, but also a tool for a deeper understanding of theoretical folding models. This study contributes to recent work that reveals the critical relevance of topology to protein folding with a new, contact-based, mathematically rigorous perspective. We show that topology can predict folding kinetics when geometry-based predictors like contact order and size fail.

Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase.

The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in the folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to 'U-shaped' DPO4 folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4-DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, which is a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes.

Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Long-range correlation in protein dynamics: Confirmation by structural data and normal mode analysis.

Proteins in cellular environments are highly susceptible. Local perturbations to any residue can be sensed by other spatially distal residues in the protein molecule, showing long-range correlations in the native dynamics of proteins. The long-range correlations of proteins contribute to many biological processes such as allostery, catalysis, and transportation. Revealing the structural origin of such long-range correlations is of great significance in understanding the design principle of biologically functional proteins. In this work, based on a large set of globular proteins determined by X-ray crystallography, by conducting normal mode analysis with the elastic network models, we demonstrate that such long-range correlations are encoded in the native topology of the proteins. To understand how native topology defines the structure and the dynamics of the proteins, we conduct scaling analysis on the size dependence of the slowest vibration mode, average path length, and modularity. Our results quantitatively describe how native proteins balance between order and disorder, showing both dense packing and fractal topology. It is suggested that the balance between stability and flexibility acts as an evolutionary constraint for proteins at different sizes. Overall, our result not only gives a new perspective bridging the protein structure and its dynamics but also reveals a universal principle in the evolution of proteins at all different sizes.

Proteomimetics as protein-inspired scaffolds with defined tertiary folding patterns.

Proteins have evolved as a variable platform that provides access to molecules with diverse shapes, sizes and functions. These features have inspired chemists for decades to seek artificial mimetics of proteins with improved or novel properties. Such work has focused primarily on small protein fragments, often isolated secondary structures; however, there has lately been a growing interest in the design of artificial molecules that mimic larger, more complex tertiary folds. In this Perspective, we define these agents as 'proteomimetics' and discuss the recent advances in the field. Proteomimetics can be divided into three categories: protein domains with side-chain functionality that alters the native linear-chain topology; protein domains in which the chemical composition of the polypeptide backbone has been partially altered; and protein-like folded architectures that are composed entirely of non-natural monomer units. We give an overview of these proteomimetic approaches and outline remaining challenges facing the field.

How to Manage Cartilage Injuries?

Although small cartilage injuries are commonly found in knee arthroscopy procedures, significant chondral and osteochondral injuries are relatively infrequent. Incidence of cartilage injury rises when considering traumatic origin, especially when approaching significant ligamentous or meniscal pathology. Options for restoration span the gamut from benign neglect to open procedures that restore both cartilage and subchondral bone. The best choice of procedure largely depends on lesion size, depth, and location. Smaller lesions isolated to cartilage <2 cm2 can be treated with marrow stimulation techniques such as microfracture with or without biologic options (bone marrow aspirate concentrate or platelet-rich plasma with or without cartilage precursors or scaffolds). Microfracture alone in larger lesions has been reported to be less durable and it is therefore not recommended for larger lesions. Smaller lesions <2 cm2 that include a subchondral injury can be treated with osteochondral autograft implantation, in which a core of cartilage and bone is transferred from a relative non-weightbearing surface to the lesion.Larger osteochondral lesions >2 cm2 are better treated with osteochondral allograft transplantation, where osteochondral cores from a size-matched, fresh cadaver are matched to the patient's lesion. This option may require multiple cores to be placed in a “snowman” pattern; however, recent literature demonstrated that a single plug might produce better outcomes. Alternatively, for large chondral-only lesions, a resurfacing procedure may be chosen that may include biologic options. Autologous chondrocyte implantation (ACI), currently in its third iteration (matrix ACI [MACI]), is an excellent choice with good long-term durability. In addition, MACI may be used for chondral lesions in the patellofemoral joint where matching the native joint topology may be more difficult. If the patient has an underlying bone marrow lesion but an intact cartilage cap that appears healthy on arthroscopic examination, one may consider a core decompression and injection with biologics such as BMAC and bony scaffold with fibrin glue (also known as bioplasty).It is also critical that the surgeon address any concomitant knee pathology that would compromise cartilage restoration. This includes addressing malalignment with distal femoral, proximal tibial, or tibial tubercle osteotomy, significant meniscal deficiency with meniscal transplant, and any instability from lack of cruciate or collateral ligaments with ligament reconstruction.

Reverse ketal-acetal rearrangement of levoglucosenone and cyclohexanone Michael adducts and the possibilities of its use in the synthesis of native topology lactones

Heating of solutions of the internal hemiketal, obtained by opening the 1,6-anhydro bridge in the Michael adduct of levoglucosenone and cyclohexanone, under reflux in THF or diglyme in the presence of NaH or Na, respectively, leads to the reduction of the hemiketal group to the ether group and oxidation of its alcohol component to the hemiacetal group. The conditions for the reverse transformation were established. Some transformations of the obtained cyclic hemiacetal have been studied. Direct methylation attempts with MeMgI in order to introduce a substituent into the 1,6-anhydro bridge were unsuccessful; therefore, the introduction of the methyl group was carried out after the oxidation of hemiacetal group to the lactone.

Searching the Optimal Folding Routes of a Complex Lasso Protein

Understanding how polypeptides can efficiently and reproducibly attain a self-entangled conformation is a compelling biophysical challenge that might shed new light on our general knowledge of protein folding. Complex lassos, namely self-entangled protein structures characterized by a covalent loop sealed by a cysteine bridge, represent an ideal test system in the framework of entangled folding. Indeed, because cysteine bridges form in oxidizing conditions, they can be used as on/off switches of the structure topology to investigate the role played by the backbone entanglement in the process. In this work, we have used molecular dynamics to simulate the folding of a complex lasso glycoprotein, granulocyte-macrophage colony-stimulating factor, modeling both reducing and oxidizing conditions. Together with a well-established Gō-like description, we have employed the elastic folder model, a coarse-grained, minimalistic representation of the polypeptide chain driven by a structure-based angular potential. The purpose of this study is to assess the kinetically optimal pathways in relation to the formation of the native topology. To this end, we have implemented an evolutionary strategy that tunes the elastic folder model potentials to maximize the folding probability within the early stages of the dynamics. The resulting protein model is capable of folding with high success rate, avoiding the kinetic traps that hamper the efficient folding in the other tested models. Employing specifically designed topological descriptors, we could observe that the selected folding routes avoid the topological bottleneck by locking the cysteine bridge after the topology is formed. These results provide valuable insights on the selection of mechanisms in self-entangled protein folding while, at the same time, the proposed methodology can complement the usage of established minimalistic models and draw useful guidelines for more detailed simulations.

Interplay between Native State Topology and Sequence in Two-State Protein Folding

Interplay between Native State Topology and Sequence in Two-State Protein Folding

Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition.

The lactose repressor, LacI (I+YQR), is an archetypal transcription factor that has been a workhorse in many synthetic genetic networks. LacI represses gene expression (apo ligand) and is induced upon binding of the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was used to confer inverted function in the native LacI topology resulting in anti-LacI (antilac) function (IAYQR), where IPTG binding results in gene suppression. Here we engineered 46 antilacs with alternate DNA binding function (IAADR). Phenotypically, IAADR transcription factors are the inverse of wild-type I+YQR function and possess alternate DNA recognition (ADR). This collection of bespoke IAADR bind orthogonally to disparate non-natural operator DNA sequences and suppress gene expression in the presence of IPTG. This new class of IAADR gene regulators were designed modularly via the systematic pairing of nine alternate allosteric regulatory cores with six alternate DNA binding domains that interact with complementary synthetic operator DNA sequences. The 46 IAADR identified in this study are also orthogonal to the naturally occurring operator O1. Finally, a demonstration of full orthogonality was achieved via the construction of synthetic genetic toggle switches composed of two nonsynonymous unit pair operations that control two distinct fluorescent outputs. This new class of IAADR transcription factors will facilitate the expansion of the computational capacity of engineered gene circuits, via the scalable increase in the control over the number of gene outputs by way of the expansion of the number of unique transcription factors (or systems of transcription factors) that can simultaneously regulate one or more promoter(s).

Research progress of molecular structure and dynamics of biological water

The specific water molecules that are confined within the solvation shell adjacent to the surface of biological macromolecules (including protein, enzyme, DNA, RNA, cell membrane, etc.) are called biological water molecules. Such water around the biomolecule surface plays a very important role in the structure, stability, dynamics, and function of biological macromolecules. A molecular-level understanding of the structure and dynamics of biological water, as well as the nature of its influence on biological structure and function is the key to revealing the mechanism of the biological functions. However, the researches in this field are still in the initial stage. Here in this paper, we review the relevant researches and recent progress of hydration water from three aspects. The first aspect is about the influence of hydration water on biological structure and function. It is evident that water actively participates in many biological processes such as protein folding, proton donation and migration, ligand binding and drug design, and allosteric effects. For example, water mediates the collapse of the chain and the search for the native topology through a funneled energy landscape. The second aspect is about the structure of water molecules around the biomolecules investigated by nuclear magnetic resonance (NMR), dielectric relaxation, neutron scattering, X-ray diffraction and ultrafast optical spectroscopy. The third aspect is about the dynamic behaviors of biological water, including the relaxation time scale, dynamic property, dynamic coupling between biomolecules and water molecules, and sub-diffusive motion of the water molecules along the protein surfaces. Different techniques measure different timescales for the motion of proteins and their hydration environment. While NMR and dielectric relaxation methods reveal the motion of biological water on a time scale from several tens of picoseconds to nanoseconds, ultrafast optical spectroscopy such as fluorescence and vibrational spectroscopy probes the hydrogen-bonding fluctuations of water on a time scale from the femtosecond to picosecond. It is therefore highly necessary to acquire a real and complete picture of the structure and dynamics of biological water by combining several different techniques. Finally, some unsolved scientific problems are also summarized in this review.

How Small-World Interactions Can Lead to Improved Quantum Annealer Designs

There are many factors that influence the design of quantum annealing processing units. Here we address the issue of improving quantum annealing processing unit designs from the point of view of the critical behavior of spin glasses. It has been argued [Phys. Rev. X 4, 021008 (2014)] that among the most difficult Ising spin-glass ground-state problems are those related to lattices which exhibit a finite-temperature spin-glass transition. Here, we show that adding small-world couplers between qubits (spins) to the native quasi-planar quantum processing unit graph results in a topology where a disordered Ising system can undergo a finite-temperature spin-glass transition, even when an Ising spin glass on the quasi-planar native graph does not display a transition into a glassy phase at any finite temperature. To ensure that these systems can be engineered with current fabrication techniques, using large-scale Monte Carlo simulations we demonstrate that highly-constrained systems restricted to a few fabrication layers and with fixed coupler angles can also exhibit a finite-temperature spin-glass transition. This indicates that these systems might be mean-field-like, which also means that embedding highly-nonplanar problems might be simplified when compared to the underlying native topology. Our results are illustrated using the quasi-planar Chimera topology currently used in the D-Wave Systems Inc. quantum annealing machines, as well as standard two-dimensional square lattices. The presented approach can be generalized to other topologies.

Implications of Molecular Topology for Nanoscale Mechanical Unfolding.

Biopolymer unfolding events are ubiquitous in biology and mechanical unfolding is an established approach to study the structure and function of biomolecules, yet whether and how mechanical unfolding processes depend on native state topology remain unexplored. Here, we investigate how the number of unfolding pathways via mechanical methods depends on the circuit topology of a folded chain, which categorizes the arrangement of intrachain contacts into parallel, crossing, and series. Three unfolding strategies, namely, threading through a pore, pulling from the ends, and pulling by threading, are compared. Considering that some contacts may be unbreakable within the relevant forces, we also study thedependence of the unfolding efficiency on the chain topology. Our analysis reveals that the number of pathways and the efficiency of unfolding are critically determined by topology in a manner that depends on the employed mechanical approach, a significant result for interpretation of the unfolding experiments.

Stereocontrolled synthesis of (9S)-ketodecanolide on the basis of Michael adducts obtained from levoglucosenone and cyclohexanone

A synthetic route was developed consisting of 10 steps for the synthesis of ketodecanolide with native topology in 11% overall yield, starting from Michael adducts of levoglucosenone and cyclohexanone.

Topology and Oligomerization of Mono- and Oligomeric Proteins Regulate Their Half-Lives in the Cell

To find additional structural constraints (besides disordered segments) that regulate protein half-lifein the cell, we herein assess the influence of nativetopology of monomeric and sequestration of oligomeric proteins into multimeric complexes in yeast,human, and mouse. Native topology acts as a molecular marker of globular protein's mechanical resistance and consequently captures their half-life variations on genome scale. Sequestration into multimeric complexes elongates oligomeric protein half-life in the cell, presumably by burying ubiquitinoylation sites and disordered segments required for proteasomal recognition. The latter effect is stronger for proteins associated with multiple complexes and for those binding early during complex self-assembly, including proteins that oligomerize with large proportions of surface buried. After gene duplication, diversification of topology and sequestration into non-identical sets of complexes alter half-lives of paralogous proteins during the course of evolution. Thus, native topology and sequestration into multimeric complexes reflect designing principles of proteins to regulate their half-lives.

Large scale genomic reorganization of topological domains at the HoxD locus

BackgroundThe transcriptional activation of HoxD genes during mammalian limb development involves dynamic interactions with two topologically associating domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior HoxD genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts.ResultsTo assess the structure–function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We assessed the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel long-range contacts. We report multi-partite associations between HoxD genes and up to three enhancers. We find that the loss of native chromatin topology leads to the remodeling of TAD structure following distinct parameters.ConclusionsOur results reveal that the recomposition of TAD architectures after large genomic re-arrangements is dependent on a boundary-selection mechanism in which CTCF mediates the gating of long-range contacts in combination with genomic distance and sequence specificity. Accordingly, the building of a recomposed TAD at this locus depends on distinct functional and constitutive parameters.

Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

Here we focus on the conformational search for the native structure when it is ruled by the hydrophobic effect and steric specificities coming from amino acids. Our main tool of investigation is a 3D lattice model provided by a ten-letter alphabet, the stereochemical model. This minimalist model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. We have three central goals here. The first one is to characterize the folding time (τ) by two distinct sampling methods, so we present two sets of 103 MC simulations for a fast protein-like sequence. The resulting sets of characteristic folding times, τ and τq were obtained by the application of the standard Metropolis algorithm (MA), as well as by an enhanced algorithm (MqA). The finding for τq shows two things: (i) the chain-solvent hydrophobic interactions {hk} plus a set of inter-residues steric constraints {ci,j} are able to emulate the conformational search for the native structure. For each one of the 103MC performed simulations, the target is always found within a finite time window; (ii) the ratio τq∕τ≅1∕10 suggests that the effect of local thermal fluctuations, encompassed by the Tsallis weight, provides to the chain an innate efficiency to escape from energetic and steric traps. We performed additional MC simulations with variations of our design rule to attest this first result, both algorithms the MA and the MqA were applied to a restricted set of targets, a physical insight is provided. Our second finding was obtained by a set of 600 independent MC simulations, only performed with the MqA applied to an extended set of 200 representative targets, our native structures. The results show how structural patterns should modulate τq, which cover four orders of magnitude; this finding is our second goal. The third, and last result, was obtained with a special kind of simulation performed with the purpose to explore a possible connection between the hydrophobic component of protein stability and the native structural topology. We simulated those same 200 targets again with the MqA, only. However, this time we evaluated the relative frequency {ϕq} in which each target visits its corresponding native structure along an appropriate simulation time. Due to the presence of the hydrophobic effect in our approach we obtained a strong correlation between the stability and the folding rate (R=0.85). So, as faster a sequence found its target, as larger is the hydrophobic component of its stability. The strong correlation fulfills our last goal. This final finding suggests that the hydrophobic effect could not be a general stabilizing factor for proteins.

Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a “trial and error” mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.

Sugar-Edge Interactions in a DNA-RNA G-Quadruplex: Evidence of Sequential C-H⋅⋅⋅O Hydrogen Bonds Contributing to RNA Quadruplex Folding.

DNA G-quadruplexes were systematically modified by single riboguanosine (rG) substitutions at anti-dG positions. Circular dichroism and NMR experiments confirmed the conservation of the native quadruplex topology for most of the DNA-RNA hybrid structures. Changes in the C8 NMR chemical shift of guanosines following rG substitution at their 3'-side within the quadruplex core strongly suggest the presence of C8-H⋅⋅⋅O hydrogen-bonding interactions with the O2' position of the C2'-endo ribonucleotide. A geometric analysis of reported high-resolution structures indicates that such interactions are a more general feature in RNA quadruplexes and may contribute to the observed preference for parallel topologies.

Temperature-induced unfolding behavior of proteins studied by tensorial elastic network model.

Motivated by single molecule experiments and recent molecular dynamics (MD) studies, we propose a simple and computationally efficient method based on a tensorial elastic network model to investigate the unfolding pathways of proteins under temperature variation. The tensorial elastic network model, which relies on the native state topology of a protein, combines the anisotropic network model, the bond bending elasticity, and the backbone twist elasticity to successfully predicts both the isotropic and anisotropic fluctuations in a manner similar to the Gaussian network model and anisotropic network model. The unfolding process is modeled by breaking the native contacts between residues one by one, and by assuming a threshold value for strain fluctuation. Using this method, we simulated the unfolding processes of four well-characterized proteins: chymotrypsin inhibitor, barnase, ubiquitein, and adenalyate kinase. We found that this step-wise process leads to two or more cooperative, first-order-like transitions between partial denaturation states. The sequence of unfolding events obtained using this method is consistent with experimental and MD studies. The results also imply that the native topology of proteins "encrypts" information regarding their unfolding process. Proteins 2016; 84:1767-1775. © 2016 Wiley Periodicals, Inc.