Modulation of folding energy landscape by charge–charge interactions: Linking experiments with computational modeling

The kinetics of folding–unfolding of a structurally diverse set of four proteins optimized for thermodynamic stability by rational redesign of surface charge–charge interactions is characterized experimentally. The folding rates are faster for designed variants compared with their wild-type proteins, whereas the unfolding rates are largely unaffected. A simple structure-based computational model, which incorporates the Debye–Hückel formalism for the electrostatics, was used and found to qualitatively recapitulate the experimental results. Analysis of the energy landscapes of the designed versus wild-type proteins indicates the differences in refolding rates may be correlated with the degree of frustration of their respective energy landscapes. Our simulations indicate that naturally occurring wild-type proteins have frustrated folding landscapes due to the surface electrostatics. Optimization of the surface electrostatics seems to remove some of that frustration, leading to enhanced formation of native-like contacts in the transition-state ensembles (TSE) and providing a less frustrated energy landscape between the unfolded and TS ensembles. Macroscopically, this results in faster folding rates. Furthermore, analyses of pairwise distances and radii of gyration suggest that the less frustrated energy landscapes for optimized variants are a result of more compact unfolded and TS ensembles. These findings from our modeling demonstrates that this simple model may be used to: (i) gain a detailed understanding of charge–charge interactions and their effects on modulating the energy landscape of protein folding and (ii) qualitatively predict the kinetic behavior of protein surface electrostatic interactions.The energy landscape theory provides a conceptual framework to describe the ensemble nature of the protein folding process (1–3). However, a more detailed understanding of contributions from specific types of interactions remains an active area of research (4, 5). Particularly, the question of how interactions between charged residues modulate the funneled energy landscape is not well explored. These interactions are long-range and thus can alter the conformational ensemble at every step of the folding process. The interactions between charged residues are also nonspecific and either attractive or repulsive and therefore their potential effects on the folding energy landscape can be highly complex (6, 7). Traditionally, the modulation of electrostatic interactions in proteins was done by changing the pH or to a lesser degree changing the ionic strength of the solution (8, 9). Such approaches are complicated by the difficulties of predicting the titration properties of individual amino acid residues in the context of ensembles of protein conformations that are sampled during the folding reaction (10). A more attractive approach is to modulate electrostatic interactions via substitutions that perturb the thermodynamic and kinetic properties of proteins using simple and computationally tractable model systems. Previously, we have shown that the stability of a diverse set of globular proteins can be modulated by rationally redesigning surface charge–charge interactions (11–26). These redesigned proteins are ideally suited to probe the role of electrostatic interactions in modulating the folding energy landscape. The redesigned variants have higher thermodynamic stability than their wild-type proteins. However, because the redesigned proteins contain very few substitutions (less than 5% of total), and because all of the substitutions are on the protein surface, they do not disrupt the native contacts that are important for defining funneled energy landscape (13, 27). Finally, the properties of these proteins can be compared at the same pH, largely eliminating the need to compute titration profiles. In this work, we used four of these redesigned proteins to experimentally probe their folding kinetics and compared them to their corresponding wild-type proteins. The experimental thermodynamic and kinetic data were further rationalized by molecular dynamics simulations using a structure-based model that incorporates the Debye–Hückel formalism to describe interactions between charges. We found that this model qualitatively predicts experimental thermodynamics and kinetics for all four studied proteins and provides insights of how charge–charge interactions modulate the protein folding energy landscape.     

Ancient dynamin segments capture early stages of host–mitochondrial integration

Eukaryotic cells use dynamins—mechano-chemical GTPases—to drive the division of endosymbiotic organelles. Here we probe early steps of mitochondrial and chloroplast endosymbiosis by tracing the evolution of dynamins. We develop a parsimony-based phylogenetic method for protein sequence reconstruction, with deep time resolution. Using this, we demonstrate that dynamins diversify through the punctuated transformation of sequence segments on the scale of secondary-structural elements. We find examples of segments that have remained essentially unchanged from the 1.8-billion-y-old last eukaryotic common ancestor to the present day. Stitching these together, we reconstruct three ancestral dynamins: The first is nearly identical to the ubiquitous mitochondrial division dynamins of extant eukaryotes, the second is partially preserved in the myxovirus-resistance-like dynamins of metazoans, and the third gives rise to the cytokinetic dynamins of amoebozoans and plants and to chloroplast division dynamins. The reconstructed sequences, combined with evolutionary models and published functional data, suggest that the ancestral mitochondrial division dynamin also mediated vesicle scission. This bifunctional protein duplicated into specialized mitochondrial and vesicle variants at least three independent times—in alveolates, green algae, and the ancestor of fungi and metazoans—accompanied by the loss of the ancient prokaryotic mitochondrial division protein FtsZ. Remarkably, many extant species that retain FtsZ also retain the predicted ancestral bifunctional dynamin. The mitochondrial division apparatus of such organisms, including amoebozoans, red algae, and stramenopiles, seems preserved in a near-primordial form.Eukaryotes arose through the acquisition of mitochondria by an archaeal host cell about 2 billion y ago (1, 2), a watershed moment in the evolution of the modern compartmentalized cell plan (3). A second transformative endosymbiotic event, the acquisition of a cyanobacterium by a eukaryotic host to form chloroplasts, gave rise to the photosynthetic eukaryotic lineages (4). As the endosymbionts became integrated with their hosts, their growth and division became regulated by host–cellular machinery (5). Proteins of the dynamin superfamily were central to this process: Mitochondria and chloroplasts originally divided using a constricting ring of the prokaryotic cytoskeletal protein FtsZ, but dynamins have been recruited to these roles in all extant eukaryotes (6, 7). By reconstructing the evolutionary history of dynamins, we can probe the process of endosymbiont integration.The dynamin superfamily is diverse (8, 9), and different dynamin variants remodel membranes at different cellular locations (Table S1 and primary references therein). A major class of dynamins is essential for mitochondrial and peroxisomal division. Another large group drives the scission of clathrin-coated vesicles in organisms such as fungi and alveolates. A related group, the so-called “classical” dynamins that drive clathrin-coated vesicle scission in metazoans and land plants, contains a membrane-targeted pleckstrin homology (PH) domain. Members of the phragmoplastin class of dynamins participate in cell plate formation in land plants. The myxovirus-resistance-like dynamins are implicated in antiviral activity in vertebrates. A truncated dynamin variant is involved in cytokinesis in amoebozoans and plants, as well as in chloroplast fission in photosynthetic lineages; another truncated variant drives mitochondrial inner membrane fusion in fungi and metazoans. Finally, mitofusins and the related bacterial dynamin-like proteins (BDLPs) are potentially ancient members of the dynamin superfamily (10); these are excluded from our study because they are highly diverged at the sequence level.Here we present the most comprehensive analysis of dynamin evolution yet reported, including thousands of functionally diverse dynamins from hundreds of broadly sampled eukaryotic species. We reconstruct the series of events that led from the primordial dynamins of the 1.8-billion-y-old last eukaryotic common ancestor (LECA) (11) to the great variety of present-day dynamins. The outcome is a nuanced picture of protein diversification, mirroring key events in the evolution of eukaryotes themselves and shedding light on the earliest stages of endosymbiont integration.     

Novel interactions of two α-Hb variants with SEA deletion α0-thalassemia: hematological and molecular analyses

Objectives: To report the hematological and molecular features as well as diagnostic aspects of the hitherto un-described interactions of two rare α-globin chain variants with α⁰-thalassemia commonly found among Southeast Asian populations.

Methods: The study was done on two adult Thai patients (P1 and P2) who had hypochromic microcytic anemia. Hb analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutations were identified by PCR and related techniques.

Results: Hb analysis of P1 using HPLC showed a normal Hb pattern, but CE demonstrated an abnormal peak at zone 7. DNA sequencing identified a CCG-CTG mutation at codon 95 of the α2 globin gene corresponding to the Hb G-Georgia [α95(G2)Pro?→?Leu(α2)] previously undescribed in the Thai population. In contrast, Hb analysis of P2 demonstrated an abnormal peak not fully separated from Hb A on HPLC, but not on CE. DNA analysis identified the rarely described Hb Nakhon Ratchasima [α63(E12)Ala?→?Val(α2)] mutation. Routine DNA analysis detected the SEA deletion α⁰-thalassemia in trans to the Hb variants in both cases. Hematological parameters were compared with those of patients with compound heterozygote for other α-globin variants and α⁰-thalassemia previously documented.

Conclusions: Identification of the patients confirmed that interaction of these rare Hb variants with α⁰-thalassemia does not lead to the Hb H disease. Differentiation of these two Hb variants from other clinically relevant hemoglobinopathies in a routine setting is, however, necessary. This can be accomplished using a combined Hb-HPLC and CE analysis followed by PCR-RFLP assays.     

Induced plant defenses,host–pathogen interactions,and forest insect outbreaks

Cyclic outbreaks of defoliating insects devastate forests, but their causes are poorly understood. Outbreak cycles are often assumed to be driven by density-dependent mortality due to natural enemies, because pathogens and predators cause high mortality and because natural-enemy models reproduce fluctuations in defoliation data. The role of induced defenses is in contrast often dismissed, because toxic effects of defenses are often weak and because induced-defense models explain defoliation data no better than natural-enemy models. Natural-enemy models, however, fail to explain gypsy moth outbreaks in North America, in which outbreaks in forests with a higher percentage of oaks have alternated between severe and mild, whereas outbreaks in forests with a lower percentage of oaks have been uniformly moderate. Here we show that this pattern can be explained by an interaction between induced defenses and a natural enemy. We experimentally induced hydrolyzable-tannin defenses in red oak, to show that induction reduces variability in a gypsy moth’s risk of baculovirus infection. Because this effect can modulate outbreak severity and because oaks are the only genus of gypsy moth host tree that can be induced, we extended a natural-enemy model to allow for spatial variability in inducibility. Our model shows alternating outbreaks in forests with a high frequency of oaks, and uniform outbreaks in forests with a low frequency of oaks, matching the data. The complexity of this effect suggests that detecting effects of induced defenses on defoliator cycles requires a combination of experiments and models.Periodic outbreaks of forest-defoliating insects severely damage valuable timber and increase atmospheric CO₂ levels by converting forests from carbon sinks to carbon sources (1). Decades of research have produced multiple hypotheses to explain defoliator outbreak cycles (2), but a decisive experiment to choose between competing hypotheses faces almost insurmountable logistical difficulties, because outbreaks occur at 5–30 y intervals and typically cover thousands of square kilometers (3). Efforts to support particular hypotheses have therefore instead relied on a mixture of observational field data, small-scale field and laboratory experiments, and mathematical models.For example, the most widely accepted hypothesis is that defoliator cycles are driven by natural enemies. Support for this hypothesis comes first of all from observational data showing that defoliators experience high rates of infection by specialist pathogens and parasitoids in peak populations (2, 4) and high rates of attack by generalist predators and parasitoids in trough populations (5). Second, experimental data have confirmed key assumptions of defoliator–natural-enemy models, and the models produce long-period, large-amplitude cycles resembling time series of insect densities and defoliation levels (6).Neither data nor models have provided meaningful support for an important alternative hypothesis, that defoliator cycles are driven by induced defenses. In many trees, antiherbivore defensive compounds increase in response to defoliation (7, 8), and such increases could in principle cause outbreaks to collapse. Direct toxic effects of induced defenses in experiments, however, are often weak, and the mechanisms underlying these defenses are often unknown or poorly understood (9). Moreover, there are no obvious signs of the effects of induced defenses in time series of defoliation or insect densities. Induced-defense models therefore provide no better an explanation for defoliator cycles than do natural-enemy models (10, 11), while additionally providing no explanation for mortality due to natural enemies. Given the successes of the natural-enemy hypothesis, these failures of the induced-defense hypothesis have led to the conclusion that induced defenses play little to no role in defoliator outbreak cycles (3). Here we argue that this conclusion is premature, by presenting evidence showing that induced defenses modulate outbreak cycles of the gypsy moth (Lymantria dispar) in North America.We suspected that induced defenses play a role in gypsy moth cycles because recent analyses of defoliation data have revealed that gypsy moth cycles differ between forest types (12). In oak–hickory (Quercus–Carya spp.) forests, in which the aboveground tree biomass is 43% oaks, severe outbreaks have alternated with mild outbreaks, leading to a strong subharmonic oscillation in time series of defoliation (Fig. 1 A and B). In oak–pine (Quercus–Pinus spp.) forests, in which the aboveground tree biomass is 15% oaks, outbreak severity has instead been roughly uniform, and there has been no subharmonic (Fig. 2 A and B). Logistic regression (12) and spatially smoothed autocorrelation (13) analyses have confirmed that these differences are statistically significant.Open in a separate windowFig. 1.Outbreak dynamics in oak–hickory forests, in data (12), and in a spatial version of an outbreak model that includes induced defenses. A and C are defoliation time series in the data and in the model, respectively, and B and D are the corresponding power spectra. For the model we show a time series based on a single realization, but to ensure that the pattern holds up over multiple realizations, the spectrum for the model is an average over 100 realizations (SI Appendix, Figs. S6–S10 shows more realizations). Because the gypsy moth is an invader and because invasion dynamics could lead to confounding effects, the data are based only on areas that were completely infested by 1975, which in practice meant mostly the New England and Mid-Atlantic sections of the United States (12).Open in a separate windowFig. 2.Outbreak dynamics in oak–pine forests. Again A and C are defoliation time series in the data and in the model, respectively, and B and D are the associated power spectra (SI Appendix, Figs. S11–S15 shows more model realizations).This difference in outbreak cycles is unlikely to be due to differences in the physical environment, because climatic conditions are effectively identical between forest types and because the soil-moisture differences that determine forest composition have no direct effect on the gypsy moth (12). Meanwhile, simple natural-enemy models that include a specialist baculovirus pathogen (14) and a generalist predator (5) can reproduce qualitative features of gypsy moth cycles (6), but standard models do not produce a subharmonic. Bjornstad et al. (13) therefore extended a natural-enemy model to allow for spatial variability in generalist-predator attack rates. Their work suggests that the subharmonic requires some kind of spatial structure, but their model only produces a subharmonic if infected larvae are allowed to disperse and uninfected larvae are not allowed to disperse. In nature both infected and uninfected larvae disperse (15), so spatial variability in generalist predators does not appear to be a sufficient explanation.We therefore considered whether the observed differences in outbreak dynamics between forest types could instead be due to differences in inducibility between genera of gypsy moth host trees. In the range of the gypsy moth in North America, defoliation induces hydrolyzable tannins in most oak species (16), including red oak (Quercus rubra) (17), black oak (Quercus velutina) (17), and chestnut oak (Quercus prinus) (18), whereas the effects of white-oak defoliation (Quercus alba) on gypsy moths are also likely due to increases in hydrolyzable tannins (19). Meanwhile, pines do not contain hydrolyzable tannins at all (20), whereas levels of hydrolyzable tannins in hickories are close to or equal to zero (21). The effects of induced hydrolyzable tannins on baculovirus transmission are therefore likely to be stronger in oak–hickory forests than in oak–pine forests because of the higher fraction of oaks in oak–hickory forests.Direct toxic effects of induced defenses on gypsy moths are known to be relatively weak (22), but like many baculoviruses (23), the gypsy moth virus is transmitted when host larvae consume virus-contaminated foliage. Induced hydrolyzable tannins in foliage can therefore alter a gypsy moth larva’s risk of infection, but as we will discuss, previous laboratory evidence for such effects (24) was not consistent with field data (14). Induced birch defenses (Betula pubescens ssp. czerepanovii) can similarly alter the responses of autumnal moth (Epirrita autumnata) larvae to artificially implanted plastic filaments in the laboratory (25), but efforts to detect induction effects on autumnal moths in the field were likewise unsuccessful. Also, there is no obvious signature of induced defenses in time series of autumnal moth defoliation (26).Accordingly, for differences in host-plant inducibility to explain the disparate dynamics of gypsy moth outbreaks in oak–hickory and oak–pine forests, induced hydrolyzable tannins in oaks must first of all affect baculovirus transmission in nature. We therefore carried out an experiment to test whether induced hydrolyzable tannins modulate baculovirus transmission under field conditions. Second, spatial variability in tree-species composition must explain the differences in outbreak dynamics between the two forest types. We therefore used a mathematical model to test whether the mechanism revealed by our field experiment produces alternating severe and mild outbreaks in simulated oak–hickory forests and consistently moderate outbreaks in oak–pine forests, as seen in the data for each forest type.     

Guest–host interactions of a rigid organic molecule in porous silica frameworks

Molecular-level interactions at organic–inorganic interfaces play crucial roles in many fields including catalysis, drug delivery, and geological mineral precipitation in the presence of organic matter. To seek insights into organic–inorganic interactions in porous framework materials, we investigated the phase evolution and energetics of confinement of a rigid organic guest, N,N,N-trimethyl-1-adamantammonium iodide (TMAAI), in inorganic porous silica frameworks (SSZ-24, MCM-41, and SBA-15) as a function of pore size (0.8 nm to 20.0 nm). We used hydrofluoric acid solution calorimetry to obtain the enthalpies of interaction between silica framework materials and TMAAI, and the values range from −56 to −177 kJ per mole of TMAAI. The phase evolution as a function of pore size was investigated by X-ray diffraction, IR, thermogravimetric differential scanning calorimetry, and solid-state NMR. The results suggest the existence of three types of inclusion depending on the pore size of the framework: single-molecule confinement in a small pore, multiple-molecule confinement/adsorption of an amorphous and possibly mobile assemblage of molecules near the pore walls, and nanocrystal confinement in the pore interior. These changes in structure probably represent equilibrium and minimize the free energy of the system for each pore size, as indicated by trends in the enthalpy of interaction and differential scanning calorimetry profiles, as well as the reversible changes in structure and mobility seen by variable temperature NMR.Knowing both the structure and molecular mobility of guest matter in nanosized pores and channels, which often differ from those in the bulk unconfined material or solution, is essential for fundamental understanding of processes in both science and technology, with applications including natural processes such as biomineralization (1–3) and membrane transport (4, 5), engineering processes such as oil recovery (6–8), CO₂ sequestration (9–11), catalysis (12–14), and biomedical processes including diagnostics and drug delivery (15–17). Most of the pioneering research has used soft matter as guests, including gas and liquid phases, low-melting point organic solids, and long-chain polymers (18–20).In our earlier studies, various calorimetric methods have been designed to investigate guest–host interactions. Piccione et al. (21) developed a novel system for hydrofluoric acid (HF) solution calorimetry to study the interactions of four different silica zeolite frameworks with several quaternary ammonium structure-directing agents (SDAs). The enthalpies of interaction were measured to be −32.0 to −181.0 kJ per mole of SDA. Slightly stronger interactions were found by Trofymluk et al. (22) for mesoporous silica phases containing long-chain molecules. Recently, Wu et al. (23) measured the enthalpy of interaction of various small molecules with mesoprous silicas using immersion calorimetry. The hydration enthalpies of a series of cation exchanged aluminosilicate or gallosilicate zeolites were studied by Sun et al. and Zhou et al. (24–27). The data suggest that water is confined energetically more tightly when Al or Ga and a charge-balancing extraframework alkali cation are present compared with pure silica.Here we take a somewhat different approach. We use a rigid, ionic, organic solid compound, N,N,N-trimethyl-1-adamantammonium iodide (TMAAI), with high melting point as guest. The TMAA⁺ cation is almost spherical and of a diameter comparable to pores in zeolites, and TMAAI is analogous to several SDAs used in zeolite synthesis (28). Our goal is to track the changes in energetics, structure, and mobility as this molecule is introduced into hosts of increasing pore size. The samples were characterized by powder X-ray diffraction (XRD), infrared spectroscopy (FTIR), and thermal analysis [thermogravimetric differential scanning calorimetry (TG-DSC)]. HF solution calorimetry has been used to investigate quantitatively the energetics of guest–host interactions of TMAAI in a series of porous silicas (SSZ-24, MCM-41, and SBA-15), with pores from 0.8 to 20.0 nm in diameter. Solid-state NMR experiments monitored changes in molecular motion upon confinement at temperature from −90 to 140 °C.     

Products of the platelet release reaction influence cardiac electrophysiology independently of platelet–platelet interactions

A novel type platelet aggregometer, a WBA Analyzer, has enabled us to obtain the platelet aggregability data immediately after blood sampling, which is considered to closely reflect in vivo platelet function. Using this analyzer, we measured the platelet aggregatory threshold index (PATI) 5 min after blood sampling and compared it with that 60 min after blood sampling in 20 healthy male volunteers (10 smokers and 10 non-smokers). In the non-smokers, PATI was 10.3 - 2.3 w M 5 min after blood sampling, and it decreased to 4.7 - 1.5 ( P <0.001) 60 min after blood sampling. In the smokers, the PATI was 7.7 - 2.9 w M 5 min after blood sampling, and it decreased to 3.8 - 1.5 ( P <0.001) at 60 min after blood sampling. In the smokers, the PATI 5 min after blood sampling increased after a 4-week cessation of smoking (10.4 - 2.9, P <0.01), although the PATI 60 min after blood sampling did not change (4.2 - 1.6 w M). The measurement of platelet aggregability immediately after blood sampling using a WBA Analyser may be useful to evaluate not only platelet function in various thrombotic disorders, but also the effects of various anti-platelet drugs. Cessation of smoking should also be encouraged in the light of the adverse effects on platelet function.     

From the Cover: Dynamics and mechanism of ultrafast water–protein interactions

Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.Water–protein interactions are critical to protein structural stability and flexibility, functional dynamics, and biological activities (1, 2). Various methods such as neutron scattering (3), NMR (4), laser spectroscopy (5, 6), and molecular dynamics (MD) simulations (7) have been used to reveal protein surface hydration and coupled water–protein dynamics on different time and length scales. Hydration water molecules often participate in various protein functions and their motions even directly “control” protein fluctuations (2, 8). Frauenfelder et al. recently proposed a unified model for protein dynamics (8): large-scale protein motions are slaved to the fluctuations of bulk solvent and controlled by solvent viscosity while internal protein motions are slaved to the fluctuations of the hydration shell and controlled by hydration water. However, direct measurements of such coupled fluctuations at physiological temperature are challenging as a result of the ultrafast nature of water motions, and therefore most studies are indirect or at low temperature (3, 4). Here, we used a tryptophan (W) scan to probe global surface hydration (9) and used femtosecond spectroscopy to follow hydration water motions and local side-chain fluctuations in real time. With temperature dependence, we systematically measured their dynamics and thus finally elucidate their ultimate relationship.     

Andersen–Tawil syndrome: Clinical and molecular aspects

Andersen–Tawil syndrome (ATS) is a rare hereditary multisystem disorder. Ventricular arrhythmias, periodic paralysis and dysmorphic features constitute the classic triad of ATS symptoms. The expressivity of these symptoms is, however, extremely variable, even within single ATS affected families, and not all ATS patients present with the full triad of symptoms. ATS patients may show a prolongation of the QT interval, which explains the classification as long QT syndrome type 7 (LQT7), and specific neurological or neurocognitive defects. In ATS type 1 (ATS1), the syndrome is associated with a loss-of-function mutation in the KCNJ2 gene, which encodes the Kir2.1 inward rectifier potassium channel. In ATS type 2 (ATS2), which does not differ from ATS1 in its clinical symptoms, the genetic defect is unknown. Consequently, ATS2 comprises all cases of ATS in which genetic testing did not reveal a mutation in KCNJ2. The loss-of-function mutations in KCNJ2 in ATS1 affect the excitability of both skeletal and cardiac muscle, which underlies the cardiac arrhythmias and periodic paralysis associated with ATS. Thus far, the molecular mechanism of the dysmorphic features is only poorly understood. In this review, we summarize the clinical symptoms, the underlying genetic and molecular defects, and the management and treatment of ATS.     

Origins of specificity and affinity in antibody–protein interactions

Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from experimental alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody–antigen complex structures indicate that aromatic, mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These aromatic side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope–epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochemical features ubiquitously distributed over all protein surfaces.It is incompletely understood as to how functional antibodies can almost always be elicited against unlimited possibilities of protein antigens from a limited repertoire of antibodies. Antibodies provide protection against foreign protein antigens by recognizing the antigen proteins with exquisite specificity and remarkable affinity, but the principles underlying the antibody affinity and specificity remain elusive. Consequently, current antibody discoveries are by and large limited by the uncontrollable animal immune systems (1) or by the recombinant antibody libraries with relatively infinitesimal coverage of the vast combinatorial sequence space in antibody–antigen interaction interfaces (2). In developing the efficacy of a therapeutic antibody, optimizing the affinity and specificity of the antibody–antigen interaction mostly relies on selecting and screening from a large pool of random candidates. As antibodies are becoming the most prominent class of protein therapeutics (3), a better understanding of the principles governing antibody affinity and specificity will facilitate in understanding humoral immunity and in developing novel antibody-based therapeutics.Antibody paratopes are enriched with aromatic residues. Tyrosyl side chains are overpopulated on the paratopes, noticeable on solving the first structures of antibody–antigen complexes (4). Surveys thereafter showed that Tyr and Trp frequently occur in the putative binding regions of antibodies as determined from structural and sequence data (5). Recent analyses of more than 100 high-resolution antibody–antigen complexes in the Protein Data Bank (PDB) confirm a similar conclusion that aromatic residues (Tyr, Trp, and Phe) are substantially enriched in antibody paratopes (6, 7). The fundamental role of the tyrosyl side chains in antibody–antigen recognition has been demonstrated (8), with the functional antibodies selected and screened from the minimalist designs of antibody complementarity determining region (CDR) libraries with only a small subset of amino acid types (Tyr, Ala, Asp, and Ser) (9) or with binary code (Tyr and Ser) (10).Interactions involving aromatic side chains on the CDRs of antibodies with epitope residues on protein antigens have been demonstrated to contribute energetically to antibody–antigen recognition. Alanine scanning of the antibody paratope residues of the FvD1.3-hen egg white lysozyme (HEL) and FvD1.3-FvE5.2 (anti-idiotype antibody) complexes and shotgun alanine scanning assessing the energetic contributions of paratope residues to VEGF and human epidermal growth factor receptor 2 (HER2) binding indicated that around half of the hot spots (ΔΔG ≥ 1 kcal/mol) are aromatic residues (20/40) (11, 12). Double-mutant cycle experiments dissecting the residue-pair coupling energies between the epitope and paratope for the two antibody–antigen complexes also indicated the predominant energetic contribution of the aromatic side chains (9/11) in the antibody–antigen interactions (13). These energetic assessments suggest that aromatic side chains contribute a substantial portion of the affinity of the antibody–antigen complexes in general. These results are consistent with the survey indicating that aromatic residues, in particular Tyr and Trp, account for a large portion of the hot spot residues in protein–protein interactions (14, 15).Aromatic side chains interact favorably with diverse functional groups in natural amino acids, underlying the affinity and specificity of the antibody–antigen recognition through a cumulative collection of relatively weak noncovalent interactions. The aromatic side chains interact with other aromatic side chains through face-to-edge or parallel π-stacking, with positively charged side chains through the cation–π interaction, with backbone and side-chain hydrogen bond donors through hydrogen bonding to aromatic π-systems (X–H–π interaction, X = N, O, S), with alkyl carbons through the C–H–π interaction, with sulfur-containing side chains through sulfur–arene interactions, and with negative charged side chains through anion–π interactions (16, 17). Although each of the above mentioned interactions is relatively weak, on the order of a few kilocalories per mole in model systems (16, 17), the cumulative sum of the interactions involving the aromatic side chains can reasonably account for the binding energy of 12 kcal/mol for a typical antibody–antigen interaction of K_d ∼1 nM at room temperature aqueous environment. Moreover, the specific preferences of the spatial geometries for the interacting functional groups involving aromatic side chains (see refs. 16–18 and references therein) also underlie the specificity of antibody–antigen recognitions. Direct hydrogen bonds bridging across the antibody–antigen interaction interface are expected to contribute to both the binding affinity and specificity, but the removal of an interface hydrogen bond is frequently inconsequential to the binding specificity and affinity due to compensating water-mediated interactions (13). These results suggest that the 3D distribution of the paratope aromatic side chains by and large determines the affinity and specificity of the antibody–antigen interaction.Known epitopes on antigens, on the other hand, are not easily distinguishable from the solvent accessible surfaces of protein structures. A recent review of the public domain conformational epitope prediction algorithms, for which the performances were compared with an independent test set for benchmarking, shows that the conformational epitope prediction problem remain challenging, with an area under the curve (AUC) ranging from 0.567 to 0.638 and accuracy from 15.5% to 25.6% (19). This moderate success rate was attributed to incomplete understanding of the essence of the epitopes (6). It has been well accepted that the solvent accessible and protruding surface regions are more likely to be conformational epitopes (20–23) and that the epitopes encompass substantially more loop residues than α-helix and β-strand residues (23, 24). By contrast, the conclusions from various studies on the amino acid composition of conformational epitopes are not consistent (6), in large part due to the fact that the epitope amino acid composition is not particularly distinguishable from the nonantigenic solvent accessible surface area (6, 7, 22, 23, 25). The contradiction has been discussed recently (25), indicating that the physicochemical complementarity between the paratopes and the epitopes are strikingly incomparable, with overwhelmingly emphasized tyrosyl side chains in all CDR loops (25).The goal of this work is to understand how a natural repertoire of antibodies, for which the sequence and structure are relatively limited in variation, can recognize protein antigens with seemly unlimited structural and sequence diversities. An extensive examination on the monoclonal antibodies elicited with a set of model antigens has concluded that a protein antigen surface consists of overlapping conformational epitopes forming a continuum; that is, no inherent property of the protein molecule could restrict antigenic site locations on the protein surface (26). It would be conceivable that antibodies recognize a common feature shared by all protein surface sites, and as such, a relatively limited population of antibodies could recognize limitless protein antigen surfaces. That is, protein surfaces are not as diverse as one would expect by looking at the protein sequences. However, this common feature on protein surface recognizable by antibodies is not known. Although aromatic side chains are known, in principle, to be able to interact favorably with wide varieties of functional groups in natural amino acids (see above), atomic details of the paratope aromatic side chains interacting favorably with diverse epitope surfaces have not been systematically analyzed. In addition, residues with short hydrophilic side chains (Ser, Thr, Asp, and Asn) are known to be enriched alongside the aromatic side chains in the paratopes (5, 24, 27), but the roles of these short hydrophilic side chains in antibody–antigen interactions have not been systematically investigated. More importantly, it has been well accepted that only hot spot residues in an antigen combining site of an antibody, i.e., the residues in the functional paratopes, are indispensable for the antibody–antigen interaction; side chains contacting the antigen (i.e., structural paratope residues) outside the functional paratope can frequently be truncated to Cβ carbon without affecting the antibody–antigen interaction (11, 13, 15, 28). To search for the relevant protein surface features recognizable by antibodies, it is desirable to first elucidate the principles governing the interactions for the functional paratopes with the corresponding functional epitopes. Such studies require a large number of well-defined functional paratopes and functional epitopes, but only a small number have been determined with the labor-intensive alanine scanning experiment (11–13, 29). To circumvent the scarcity of the experimental data, we use computational methods to predict the functional paratopes/epitopes in antibody–protein complex structures so that the key interactions involving the hot spots in the antibody–protein interactions can be elucidated, at least to the reliable extent depending on the functional paratope prediction accuracy.In this work, we applied computational methods to predict functional paratopes on antibody variable domains and analyzed the key atomistic contact pairs in the functional paratope–epitope interfaces. Although the structural paratope–epitope interfaces can be defined from the known complex structures, the functional binding interfaces involving hot spot residues are unknown experimentally and need to be defined with computational predictions. One set of predictions was carried out with our previously published computational method [In-silico Molecular Biology Lab–protein-protein interaction (ISMBLab-PPI)], where the protein–protein interaction confidence level (PPI_CL) for protein surface atoms to participate in protein–protein interaction is strongly correlated (r² = 0.99) with the averaged burial level of the atoms in the PPI interfaces (30). Another set of predictions was carried out with a recently published random forest algorithm, prediction of antibody contacts (proABC) (31), which was trained specifically with antibody–antigen complex structures in PDB with additional information from antibody germ-line family sequences, CDR residue positions, multiple antibody sequence alignments, CDR lengths and canonical structures, and antigen volume. Both sets of predicted functional paratope–epitope interfaces consistently led to the conclusion that antibodies, with relatively limited sequence and structural diversities in the antigen binding sites, can recognize unlimited protein antigens through recognizing the common and ubiquitous physicochemical features on all protein surfaces. The implication is that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces.     

Subclinical infections of cardiac implantable electronic devices: Insights into the host–bacteria dialog from blood and pocket tissue with pyrosequencing

While bacteria exist in CIED patients without clinical signs of infection, the underlying bacterial community structure and diversity in the bloodstream and pocket tissue of asymptomatic CIED patients remain unknown. In this study, we performed high-throughput 454 pyrosequencing of bacterial 16S rDNA of blood and pocket tissue from 54 asymptomatic CIED patients as well as blood from 30 normal individuals (normal controls). Firstly, we observed a significant increase of blood bacterial diversity in patients as compared with blood of normal subjects or patient tissues. We also found significant differences in 13 blood-associated bacterial genera between patients and normal subjects, and 14 bacteria genera between blood and tissues within patients. Secondly, we found that the serum levels of four inflammatory markers (CRP, IL-1β, IL-6, and MCP-1) in CIED patients were significantly higher than those in normal subjects. Thirdly, we found that there were significant correlations between 43 bacterial species and these inflammatory markers. Taken together, our results reveal a high diversity in the microbial community in CIED patients, and suggest the potential roles of multiple bacteria co-occurrence in the CIED subclinical infections.     

A low molecular weight,heat-labile factor enhances neutrophil fc receptor–mediated lysosomal enzyme release and phagocytosis

When exposed to solid-phase immune complexes, polymorphonuclear neutrophils (PMN) degranulate and release proteases capable of degrading the major structural macromolecules of the joint. Evidence indicates that the PMN response to such activators may be modified by factors present at the sites of inflammation. We have evaluated the effects of a low molecular weight factor present in synovial fluid from rheumatoid arthritis (RA) patients on Fc receptor-mediated PMN degranulation and phagocytosis. Synovial fluid samples from 11 RA patients were studied; 10 of them contained factor(s) which augmented phagocytosis and degranulation mediated through the Fc receptor. This factor(s) alone, however, did not initiate neutrophil degranulation: its MW is less than 10,000 daltons and it is unstable when heated to 56°C. We also examined supernatants that were produced by coculturing adherent human mononuclear cells stimulated by IgG-coated sheep red blood cells with autologous nonadherent mononuclear cells. A factor(s) with properties similar to those found in the synovial fluids (i.e., heat-labile at 56°C and capable of augmenting Fc receptor-mediated degranulation and phagocytosis) was found in the stimulated human mononuclear cell culture supernatants. Filtration studies indicated that the MW of this factor(s) is between 2,000 and 10,000 daltons. No such activity was detected in culture supernatants of unstimulated mononuclear cells. Production of the cytokine was blocked by cycloheximide, indicating that protein synthesis was necessary. This factor(s), by enhancing PMN degranulation, may augment PMN-mediated tissue destruction in diseases such as RA.     

Observation of femtosecond X-ray interactions with matter using an X-ray–X-ray pump–probe scheme

Resolution in the X-ray structure determination of noncrystalline samples has been limited to several tens of nanometers, because deep X-ray irradiation required for enhanced resolution causes radiation damage to samples. However, theoretical studies predict that the femtosecond (fs) durations of X-ray free-electron laser (XFEL) pulses make it possible to record scattering signals before the initiation of X-ray damage processes; thus, an ultraintense X-ray beam can be used beyond the conventional limit of radiation dose. Here, we verify this scenario by directly observing femtosecond X-ray damage processes in diamond irradiated with extraordinarily intense (∼10¹⁹ W/cm²) XFEL pulses. An X-ray pump–probe diffraction scheme was developed in this study; tightly focused double–5-fs XFEL pulses with time separations ranging from sub-fs to 80 fs were used to excite (i.e., pump) the diamond and characterize (i.e., probe) the temporal changes of the crystalline structures through Bragg reflection. It was found that the pump and probe diffraction intensities remain almost constant for shorter time separations of the double pulse, whereas the probe diffraction intensities decreased after 20 fs following pump pulse irradiation due to the X-ray–induced atomic displacement. This result indicates that sub-10-fs XFEL pulses enable conductions of damageless structural determinations and supports the validity of the theoretical predictions of ultraintense X-ray–matter interactions. The X-ray pump–probe scheme demonstrated here would be effective for understanding ultraintense X-ray–matter interactions, which will greatly stimulate advanced XFEL applications, such as atomic structure determination of a single molecule and generation of exotic matters with high energy densities.Since W. C. Röntgen discovered X-rays emitted from vacuum tube equipment in 1895, scientists have continuously endeavored to develop brighter X-ray sources throughout the 20th century. One of the most remarkable breakthroughs was the emergence of synchrotron light sources, which were much more brilliant than the early lab-based X-ray sources. Such dramatic increase in X-ray brilliance provided a pathway to obtain high-quality X-ray scattering data. This, in turn, enabled one to solve the structures of complex systems such as proteins, functional units of living organisms, and viruses. However, the increase in the brilliance is also accompanied by a severe problem of X-ray radiation damage to the samples being examined (1). X-rays ionize atoms and generate highly activated radicals that break chemical bonds and cause changes in the structures of the samples. To achieve structure determination precisely, a sufficient scattering signal should be recorded before the samples are severely damaged. Radiation damage was considered to be an intrinsic problem associated with X-ray scattering experiments, which imposed a fundamental limit on the resolution in X-ray structure determination (2).The recent advent of X-ray free-electron lasers (XFELs) (3–5), which emit ultraintense X-ray pulses with durations of several femtoseconds, may totally avoid the problem of radiation damage. The irradiation of intense XFEL pulses generates highly ionized atoms, and the strong Coulomb repulsive force leads to evaporation of the samples. Meanwhile, it has been predicted theoretically (6) that atoms do not change their positions before the termination of the femtosecond X-ray pulse owing to inertia, thus enabling the use of X-ray radiations beyond the conventional X-ray dose limit. This innovative concept, called a “diffraction-before-destruction” scheme (6, 7), has paved a clear way to high-resolution structure determinations of weak scattering objects, including nanometer-sized protein crystals (8), noncrystalline biological particles (9), and damage-sensitive protein crystals (10).Despite the potential impact of XFELs, detailed understanding of the ultrafast XFEL damage processes has been missing. As a pioneering work, Barty et al. (11) measured the diffraction intensities of protein nanocrystals by changing the XFEL pulse durations from 70 to 300 fs at intensities of ∼10¹⁷ W/cm². They found that the diffraction intensities greatly decrease for longer durations, clearly indicating sign of structural damage, i.e., X-ray–induced atomic displacements within the XFEL pulse durations. For further understanding of ultraintense X-ray interactions with matter, we need to directly measure the temporal changes of the structural damage. In particular, measuring the ignition time of the atomic displacements is crucial for realizing advanced applications with greatly intense XFELs. Although improving our knowledge of the X-ray damage processes is essential for all aspects of XFEL science, the experimental verifications have been missing because of the extreme difficulty in observation with ultrahigh resolutions in space (ångstrom) and time (femtosecond).As a new approach to investigate the femtosecond X-ray damage processes, we here propose an X-ray–X-ray pump–probe experiment using double X-ray pulses; a pump X-ray pulse excites a sample and a probe X-ray pulse with a well-controlled time delay characterizes the change in the sample. In this approach, it is highly useful to exploit two-color double pulses with tunable temporal separations (12–15), which have been developed at SPring-8 Angstrom Compact free-electron LAser (SACLA) (4) and Linac Coherent Light Source (3). In this article, we measured the X-ray damage processes of diamond by using an X-ray–X-ray pump–probe diffraction experiment at SACLA. As the carbon–carbon bond is one of the most fundamental bonds in biomolecules, our results should provide a benchmark for XFEL-induced damage to practical samples.     

Single-molecule imaging and kinetic analysis of cooperative cofilin–actin filament interactions

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a single-molecule imaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 µM⁻¹⋅s⁻¹ when the cofilin concentration was in the range of 30 nM to 1 µM. A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 0.6 s⁻¹. During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.Cofilin (1, 2) induces actin disassembly by severing actin filaments at concentrations below the equilibrium dissociation constant, whereas it facilitates actin nucleation at higher concentrations (3). This concentration-dependent modulation of cofilin activity is presumably due to its cooperative binding to actin filaments (4). In the wider sense, understanding the cooperative interactions between proteins is essential for understanding the regulation of enzymes (5, 6). However, the dynamic protein interactions that endow proteins with cooperativity in solution have not been directly imaged and analyzed. In this study, we focused on the molecular mechanism underlying the cooperative binding of cofilin to actin filaments using high-resolution optical microscopy.There is a general agreement that the binding of cofilin to actin filaments changes the conformation of the actin filaments. Electron microscopic observations reveal that the binding of cofilin to actin filaments increases their twist (4, 7). Biochemical analyses of the kinetics of cofilin binding to actin filaments suggest that the conformational changes induced by cofilin binding affect further binding of cofilin to the actin filament (8). Differential scanning calorimetric study of cofilin–actin complexes indicates that the allosteric destabilization of the actin filament by cofilin binding is propagated over a long distance (128 subunits) along the actin filament (9, 10). Electron microscopic studies show that the actin filaments are either fully decorated with cofilin or bare in the same micrograph (4, 7), suggesting that the binding of cofilin to the actin filament is highly cooperative. Related to these studies, bound cofilin allosterically accelerates Pi release from unoccupied subunits, and the acceleration of Pi release is propagated allosterically from cofilin-occupied sites to more than 10 vacant subunits along the filaments (11). The effect of bound cofilin on the conformation and dynamics of actin filaments was also examined with time-resolved phosphorescence anisotropy, which suggests that a cofilin-mediated effect (i.e., lowering the torsional rigidity) can be propagated to ca. 90 vacant subunits in the filament (12).In contrast, biochemical analyses of the cofilin–actin filament binding, assuming the nearest neighbor cooperativity model, indicate that the binding cooperativity is not so strong, and the predicted cofilin cluster size for filament severing is small (a few subunits) (3, 8, 13, 14). As noted previously (8, 13, 14), the low cooperativity precludes distinguishing between nearest neighbor and nonnearest neighbor cooperative binding models from existing data acquired by ensemble methods. The nearest neighbor cooperativity model (13) can, in theory, be directly tested by observing individual cofilin molecules binding to actin filaments in solution. This has not been conducted because the spatial resolution of the conventional optical microscope is limited (ca. 300 nm), and the precise measurement of the time course of a single cofilin binding to an actin filament in solution requires a great deal of skill.In this study, we have developed a very low background fluorescence imaging technique and addressed the fundamental, unresolved issue regarding cofilin binding and cooperative interaction with actin filaments by performing real-time single-molecule imaging of cofilin–actin filament interactions. The binding and dissociation of single cofilin molecules to and from the actin filaments were directly observed, and the spatial properties of the cooperative binding were examined for the first time to our knowledge using super-high-resolution imaging techniques (15, 16); the position of the fluorophore at the center of the image can be estimated by calculating the centroid of the fluorescence image, which allows localization to a precision about an order of magnitude greater than the microscope resolution, and is named super-high-resolution imaging (16). Quantitative analyses of the results showed that a single cofilin binding event enhanced additional binding events within ∼65 nm of the initial binding site, but the off-rate was not apparently affected. Thus, cofilin cooperativity propagated across 24 actin subunits. The molecular mechanism behind this cooperative binding is discussed on the basis of the kinetics data.     

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein–protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t_1/2 = 5.1 h and t_1/2 = 4.4 h, respectively) compared to WT-AQP2 (t_1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.