Complex Folding Research Articles

A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions

A major goal of modern protein chemistry is to create new proteins with different functions. One approach is to amalgamate secondary and tertiary structures from different proteins. This is difficult for several reasons, not the least of which is the fact that the junctions between secondary and tertiary structures are not degenerate and usually affect the function and folding of the entire complex. Here, we offer a solution to this problem by coupling a large combinatorial library of about 10(7) different N- and C-terminal junctions to a powerful system that selects for function. Using this approach, the entire Leptin and follicle-stimulating hormone (FSH) were inserted into an antibody. Complexes with full retention of function invivo and invitro, although rare, were found easily by using an autocrine selection system to search for hormonal activity. Such large diversity systems, when coupled to robust selection systems, should enable construction of novel therapeutic proteins.

Surface-Based Morphometric Analysis of Hippocampal Subfields in Mild Cognitive Impairment and Alzheimer's Disease.

The hippocampus is widely studied with neuroimaging techniques given its importance in learning and memory and its potential as a biomarker for Alzheimer's disease (AD). Its complex folding anatomy often presents analytical challenges. In particular, the critical subfield information is typically not addressed by the existing hippocampal shape studies. To bridge this gap, we present a computational framework for surface-based morphometric analysis of hippocampal subfields. The major strengths of this framework are as follows: (a) it performs detailed hippocampal shape analysis, (b) it embraces, rather than ignores, the important hippocampal subfield information, and (c) it analyzes regular magnetic resonance imaging scans and is applicable to large scale studies. We demonstrate its effectiveness by applying it to the identification of regional hippocampal subfield atrophy patterns associated with mild cognitive impairment and AD.

Chapter 5 Frontier onshore petroleum basins of Myanmar

The Myanmar Ministry of Energy has identified as prospective the three basins discussed here, but they have seen little exploration activity. Arguably the Hukawng Basin is distinct from the others insofar as exploration drilling has been carried out, and a potentially prospective stratigraphic succession has been encountered. Moreover, this basin lies on trend with the petroliferous basins of the Central Myanmar Depression. But it would be hard to argue that it is anything other than a frontier basin, and for that reason it is included here. For various reasons we consider the other basins discussed here to carry higher risk. The Ministry's onshore map accompanying the details of the 2013 bidding round included two blocks on the western flank of the Shan Plateau in what MOGE calls the Kalaw Basin: Blocks PSC-U and PSC-V. These blocks, which lie NE and east of the capital Naypyidaw (see Chapter 4, Fig. 4.2), were not however included in their list of blocks on offer. It is an area of complex folding (e.g. Garson et al. 1976), much affected by NNW–SSE faults of which some (such as the Panlaung Fault) are considered by Ridd & Watkinson (2013) to have had a history of dextral strike-slip movement during the Late Cretaceous–Palaeogene. The western part of the two blocks (i.e. west of the Panlaung Fault) exposes Palaeozoic sediments of the Slate Belt intruded by granites (Mitchell et al. 2007), and is considered to have no petroleum potential. The eastern part of the blocks is more difficult to assess. Above the ubiquitous Permian–Triassic Plateau Limestone (=Shan Dolomite), Triassic flysch (Shweminbon Formation) passes up into non-marine younger Mesozoic rocks including red beds and coal-bearing intervals. We are not aware of any petroleum exploration having taken place, and any potential this area may have is considered to be high …

Late Paleozoic evolution of the South Tien Shan: Insights from P–T estimates and allanite geochronology on retrogressed eclogites (Chatkal range, Kyrgyzstan)

In the South Tien Shan range (Kyrgyzstan), the Late Paleozoic geodynamic evolution remains debated especially to the west of the Talas-Fergana fault (TFF) fault where suture-related high-pressure (HP) rocks are scarce. We provide new petrological and geochronological data on garnet amphibolites from the Chatkal range, to the west of the TFF, northwest of the South Tien Shan suture. These rocks are retrogressed eclogites. We used a micro-mapping approach combined with forward modeling and empirical thermobarometry to decipher the P–T path of these amphibolitized eclogites. The metamorphic peak conditions culminated at 490±50°C and 18.5±2kbar and were followed by higher temperature retrogression (∼560°C at 11–7kbar). In order to constrain the age of the HP stage, we dated allanite crystals texturally coeval to the HP mineral assemblage. Allanite grains dated in situ with a U–Pb LA-ICPMS methodology yield an age of 301±15Ma. Compared with previously published data for the east of the TFF, these P–T constraints allow improving the understanding of the Late Paleozoic geodynamic evolution of the South Tien Shan. To the east of TFF, the Turkestan Ocean closed around 320Ma with the collision of the Tarim Craton with the Kazakh microcontinent. To the west of TFF, the Turkestan Ocean closed around 300Ma, when the Alai block collided with the Kazakh microcontinent. This later collision involved nappe-stacking and intense subvertical folding in the western South Tien Shan. This complex folding explains the S-shape of the suture to the west of the TFF that cannot be observed in the eastern part. These new data allow us to propose a distinct tectonic evolution of the two sides of the TFF, which suggests that this fault was a major transform fault before being a strike-slip intra-continental fault.

Did the Kyrenia Range of northern Cyprus rotate with the Troodos–Hatay microplate during the tectonic evolution of the eastern Mediterranean?

Previous palaeomagnetic studies have allowed the recognition of a distinctive area of Neotethyan oceanic rocks, including the Troodos ophiolite in Cyprus and the Hatay ophiolite to the east in southern Turkey, that underwent 90° of anticlockwise rotation between Late Cretaceous (Campanian) and Early Eocene time. The southern and western boundaries of this rotated Troodos–Hatay microplate have been inferred to lie within, or adjacent to, zones of deformed oceanic and continental margin rocks that are now exposed in southern and western Cyprus; however, the northern boundary of the microplate remains undefined. Relevant to this problem, palaeomagnetic data are presented here from basaltic lavas exposed along the Kyrenia Range, mostly from Late Cretaceous (Maastrichtian) sites and one Eocene site. A positive inclination-only fold test demonstrates that remanences are pre-deformational in age, and positive conglomerate tests show that magnetic remanences were acquired before Late Eocene–Early Oligocene time, together suggesting that primary magnetizations are preserved. Data from the eastern Kyrenia Range and the Karpas Peninsula (the easternmost extension of the Kyrenia Range) document significant relative tectonic rotation between these localities, with no rotation in the eastern range versus 30° of anticlockwise rotation of the Karpas Peninsula. Unfortunately, palaeomagnetic sites from the western Kyrenia Range did not yield tectonically interpretable magnetization directions, probably due to complex poly-phase thrusting and folding, and the central range also yielded no interpretable data. However, the available palaeomagnetic data are sufficient to demonstrate that the Kyrenia terrane underwent a separate rotation history to the Troodos–Hatay microplate and also implies that the northern boundary of the Troodos–Hatay microplate was located between the Troodos ophiolite and the Kyrenia Range. The former microplate margin has since been overridden and concealed by two phases of southwards thrusting and folding of the Kyrenia Range units (Mid-Eocene; latest Miocene–earliest Pliocene). The likely cause of the anticlockwise rotation affecting the Karpas Peninsula, and by implication the curvature of the Kyrenia Range as a whole, relates to regional late-stage subduction and diachronous continental collision. The Southern Neotethys sutured in SE Turkey during the Early Miocene, whereas a relict ocean basin remained further west in the easternmost Mediterranean, allowing a remnant N-dipping subduction zone to retreat southwards and so induce the present-day arcuate shape of the Kyrenia Range.

Involvement of Long‐Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G‐Quadruplex

AbstractThe energy landscapes of human telomeric G‐quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real‐time NMR spectroscopy, we investigated the K+‐induced folding of the human telomeric DNA sequence 5′‐TTGGG(TTAGGG)3A‐3′. Three long‐lived states were detected during folding: a major conformation (hybrid‐1), a previously structurally uncharacterized minor conformation (hybrid‐2), and a partially unfolded state. The minor hybrid‐2 conformation is formed faster than the more stable hybrid‐1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin‐like structures.

Involvement of Long-Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G-Quadruplex.

The energy landscapes of human telomeric G-quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real-time NMR spectroscopy, we investigated the K(+)-induced folding of the human telomeric DNA sequence 5'-TTGGG(TTAGGG)3 A-3'. Three long-lived states were detected during folding: a major conformation (hybrid-1), a previously structurally uncharacterized minor conformation (hybrid-2), and a partially unfolded state. The minor hybrid-2 conformation is formed faster than the more stable hybrid-1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin-like structures.

Marginally hydrophobic transmembrane α-helices shaping membrane protein folding.

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment.

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.

Twenty years of RNA crystallography.

Twenty years of RNA crystallography.

RNA dances to center stage.

RNA dances to center stage.

Reduced alphabet for protein folding prediction.

What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28-letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28-letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design.

Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR.

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function.

Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy on the Folding Mechanism of B Domain of Protein A

Molecular-level description of the folding process of proteins is of fundamental importance for understanding how proteins acquire their unique conformations to show various biological functions. Elucidation of the folding mechanism requires quantitative analysis with a high time-resolution. To this aim, we recently developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)1,2. This method enables us to examine the microsecond conformational dynamics of proteins at the single-molecule level, through the analysis of the fluorescence lifetime correlation. In this study, we applied 2D FLCS to study the folding mechanism of B domain of protein A (BdpA). This small protein was believed to show a two-state folding process in which the native state unfolds without exhibiting any intermediate states. However, a recent single-molecule study suggested that the folding mechanism of BdpA is not so simple3. To quantitatively analyze the complex folding process of BdpA, we performed 2D FLCS for two FRET mutants of BdpA. The results showed that the conformations of both the native and unfolded states are highly heterogeneous and that the conformational dynamics within each conformational ensemble occurs on a time scale shorter than ten microseconds. Furthermore, our results indicated that the conformational distribution in the native and unfolded ensembles gradually change with the change of the denaturant concentration.[1] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11414-11422.[2] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11423-11432.[3] Oikawa et al., 2013. Sci. Rep. 3, 2151.

Abnormal asymmetry of brain connectivity in schizophrenia.

Recently, a growing body of data has revealed that beyond a dysfunction of connectivity among different brain areas in schizophrenia patients (SCZ), there is also an abnormal asymmetry of functional connectivity compared with healthy subjects. The loss of the cerebral torque and the abnormalities of gyrification, with an increased or more complex cortical folding in the right hemisphere may provide an anatomical basis for such aberrant connectivity in SCZ. Furthermore, diffusion tensor imaging studies have shown a significant reduction of leftward asymmetry in some key white-matter tracts in SCZ. In this paper, we review the studies that investigated both structural brain asymmetry and asymmetry of functional connectivity in healthy subjects and SCZ. From an analysis of the existing literature on this topic, we can hypothesize an overall generally attenuated asymmetry of functional connectivity in SCZ compared to healthy controls. Such attenuated asymmetry increases with the duration of the disease and correlates with psychotic symptoms. Finally, we hypothesize that structural deficits across the corpus callosum may contribute to the abnormal asymmetry of intra-hemispheric connectivity in schizophrenia.

Rise-time of FRET-acceptor fluorescence tracks protein folding.

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway.

Uncovering the Role of Sgf73 in Maintaining SAGA Deubiquitinating Module Structure and Activity

The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex performs multiple functions in transcription activation including deubiquitinating histone H2B, which is mediated by a subcomplex called the deubiquitinating module (DUBm). The yeast DUBm comprises a catalytic subunit, Ubp8, and three additional subunits, Sgf11, Sus1 and Sgf73, all of which are required for DUBm activity. A portion of the non-globular Sgf73 subunit lies between the Ubp8 catalytic domain and the ZnF-UBP domain and has been proposed to contribute to deubiquitinating activity by maintaining the catalytic domain in an active conformation. We report structural and solution studies of the DUBm containing two different Sgf73 point mutations that disrupt deubiquitinating activity. We find that the Sgf73 mutations abrogate deubiquitinating activity by impacting the Ubp8 ubiquitin-binding fingers region and they have an unexpected effect on the overall folding and stability of the DUBm complex. Taken together, our data suggest a role for Sgf73 in maintaining both the organization and the ubiquitin-binding conformation of Ubp8, thereby contributing to overall DUBm activity.

Traveling slippery patches produce thickness‐scale folds in ice sheets

AbstractLarge, complex stratigraphic folds that rise as high as 60% of the local ice thickness have been observed in ice sheets on Antarctica and Greenland. Here we show that ice deformation caused by heterogeneous and time‐variable basal sliding can produce the observed structures. We do this using a thermomechanical ice sheet model in which sliding occurs when the base approaches the melting point and slippery patches develop. These slippery patches emerge and travel downstream because of a feedback between ice deformation, vertical flow, and temperature. Our model produces the largest overturned structures, comparable to observations, when the patches move at about the ice column velocity. We conclude that the history of basal slip conditions is recorded in the ice sheet strata. These basal conditions appear to be dynamic and heterogeneous even in the slow‐flowing interior regions of large ice sheets.

A new window into the molecular physiology of membrane proteins.

Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome.

Effect of hydrophobic interactions on the folding mechanism of β-hairpins.

Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTWENGKWTWK-NH2) that is stabilized by two cross-strand Trp-Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process.