The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.     

A systematic study of the solid state and solution phase conformational preferences of β-peptides derived from transpentacin

The solid state and solution phase conformational preferences of a homologous series of β-peptides derived from (S,S)-2-aminocyclopentanecarboxylic acid (transpentacin) have been investigated using a variety of spectroscopic and crystallographic techniques. These studies indicate that the hexamer and pentamer persist as a 12-helix in both the solid state and solution phase. Although the conformational traits of a 12-helix are exhibited by oligomers with as few as three residues in the solid state, in solution the trimer exists as an equilibrium of many alternative conformers whilst the tetramer has been shown to predominantly exist in either a 12-helix or a turn-type conformation.     

Revealing time bunching effect in single-molecule enzyme conformational dynamics

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.     

A sequence-based computational model for the prediction of the solvent accessible surface area for α-helix and β-barrel transmembrane residues

Predicting the solvent accessible surface area (ASA) of transmembrane (TM) residues is of great importance for experimental researchers to elucidate diverse physiological processes. TM residues fall into two major structural classes (α-helix membrane protein and β-barrel membrane protein). The reported solvent ASA prediction models were developed for these two types of TM residues respectively. However, this prevents the general use of these methods because one cannot determine which model is suitable for a given TM residue without information of its type. To conquer this limitation, we developed a new computational model that can be used for predicting the ASA of both TM α-helix and β-barrel residues. The model was developed from 78 α-helix membrane protein chains and 24 β-barrel membrane protein. Its prediction ability was evaluated by cross validation method and its prediction result on an independent test set of 20 membrane protein chains. The results show that our model performs well for both types of TM residues and outperforms other prediction model which was developed for the specific type of TM residues. The prediction results also proved that the random forest model incorporating conservation score is an effective sequence-based computational approach for predicting the solvent ASA of TM residues.     

Unfolding and refolding details of lysozyme in the presence of β-casein micelles

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.     

Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.     

Self-association-driven transition of the β-peptidic H12 helix to the H18 helix

Various patterns of foldameric oligomers formed by trans-ABHC ((1S,2S,3S,5S)-2-amino-6,6-dimethylbicyclo[3.3.1]heptane-3-carboxylic acid) and β(3)-hSer residues were studied. NMR, ECD and molecular modelling demonstrated that octameric and nonameric sequences with multiple i-i+3 ABHC pair repulsions attain the β-H18 helix in CD(3)OH. As a close relative of the α-helix, this helix type is stabilized by i-i+4 backbone H-bond interactions. The formation of the β-H18 helix was found to be solvent- and concentration-dependent. Upon dilution, the β-H18 → β-H12 helix transition was revealed by concentration-dependent ECD, DOSY-NMR and TEM measurements.     

A multi-functional peptide as an HIV-1 entry inhibitor based on self-concentration, recognition, and covalent attachment

HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. The C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. A conserved salt bridge between Lys(574) in NHR and Asp(632) in CHR plays an essential role in the formation of the six-helix bundle. A multi-functional peptide inhibitor for anti-HIV derived from the CHR of gp41 has been designed. It bears a cholesterol group (Chol) at the C-terminal through which the inhibitor can anchor in the cell membrane, and carries an isothiocyanate (NCS) group at the side chain of Asp(632) through which the inhibitor can bind to target covalently at Lys(574) in NHR. The dual functionalized peptide (NCS-C34-Chol) shows high antiviral activity in vitro and in vivo. The inhibitor reacts specifically and rapidly to NHR from gp41. In addition, it exhibits better stability under the digestion of the Proteinase K than C34 and T20.     

Study of early stages of amyloid Aβ13-23 formation using molecular dynamics simulation in implicit environments

β-amyloid aggregation and formation of senile plaques is one of the hallmarks of Alzheimer’s disease (AD). It leads to degeneration of neurons and decline of cognitive functions. The most aggregative and toxic form of β-amyloid is Aβ_1-42 but in experiments, the shorter forms able to form aggregates are also used. The early stages of amyloid formation are of special interest due to the influence of this peptide on progression of AD. Here, we employed nine helices of undecapeptide Aβ_13-23 and studied progress of amyloid formation using 500 ns molecular dynamics simulation and implicit membrane environment. The small β-sheets emerged very early during simulation as separated two-strand structures and a presence of the membrane facilitated this process. Later, the larger β-sheets were formed. However, the ninth helix which did not form paired structure stayed unchanged till the end of MD simulation. Paired helix–helix interactions seemed to be a driving force of β-sheet formation at early stages of amyloid formation. Contrary, the specific interactions between α-helix and β-sheet can be very stable and be stabilized by the membrane.     

Predicting three-dimensional structures of transmembrane domains of β-barrel membrane proteins

β-Barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They are important for pore formation, membrane anchoring, and enzyme activity. These proteins are also often responsible for bacterial virulence. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank. We have developed a computational method for predicting structures of the transmembrane (TM) domains of β-barrel membrane proteins. Based on physical principles, our method can predict structures of the TM domain of β-barrel membrane proteins of novel topology, including those from eukaryotic mitochondria. Our method is based on a model of physical interactions, a discrete conformational state space, an empirical potential function, as well as a model to account for interstrand loop entropy. We are able to construct three-dimensional atomic structure of the TM domains from sequences for a set of 23 nonhomologous proteins (resolution 1.8-3.0 ?). The median rmsd of TM domains containing 75-222 residues between predicted and measured structures is 3.9 ? for main chain atoms. In addition, stability determinants and protein-protein interaction sites can be predicted. Such predictions on eukaryotic mitochondria outer membrane protein Tom40 and VDAC are confirmed by independent mutagenesis and chemical cross-linking studies. These results suggest that our model captures key components of the organization principles of β-barrel membrane protein assembly.     

Effects of amphiphile topology on the aggregation of oligocholates in lipid membranes: macrocyclic versus linear amphiphiles

A macrocyclic and a linear trimer of a facially amphiphilic cholate building block were labeled with a fluorescent dansyl group. The environmentally sensitive fluorophore enabled the aggregation of the two oligocholates in lipid membranes to be studied by fluorescence spectroscopy. Concentration-dependent emission wavelength and intensity revealed a higher concentration of water for the cyclic compound. Both compounds were shown by the red-edge excitation shift (REES) to be located near the membrane/water interface at low concentrations, but the cyclic trimer was better able to migrate into the hydrophobic core of the membrane than the linear trimer. Fluorescent quenching by a water-soluble (NaI) and a lipid-soluble (TEMPO) quencher indicated that the cyclic trimer penetrated into the hydrophobic region of the membrane more readily than the linear trimer, which preferred to stay close to the membrane surface. The fluorescent data corroborated with the previous leakage assays that suggested the stacking of the macrocyclic cholate trimer into transmembrane nanopores, driven by the strong associative interactions of water molecules inside the macrocycles in a nonpolar environment.     

The roles of aldehyde dehydrogenases (ALDHs) in the PDH bypass of Arabidopsis

Background

Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of α-helix 5 of the Bacillus thuringiensisCry1Ab δ-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane.

Results

Amino acid residue leucine 157, located in the N-terminal portion of α-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the α-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the α-helix 5 is shown to be involved in ion transport activity.

Conclusion

Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the α-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire α-helix becomes inserted in the insect midgut membrane.     

Protein stability at a carbon nanotube interface

The interactions of proteins with solid surfaces occur in a variety of situations. Motivated by the many nanoengineering applications of protein-carbon nanotube hybrids, we investigate the conformational transitions of hen egg white lysozyme adsorbed on a carbon nanotube. Using a C(α) structure-based model and replica exchange molecular dynamics, we show how the folding/unfolding equilibrium of the adsorbed protein varies with the strength of its coupling to the surface. The stability of the native state depends on the balance between the favorable entropy and unfavorable enthalpy change on adsorption. In the case of a weakly attractive surface when the former dominates, the protein is stabilized. In this regime, the protein can fold and unfold while maintaining the same binding fraction. With increasing surface attraction, the unfavorable enthalpic effect dominates, the native state is destabilized, and the protein has to extensively unbind before changing states from unfolded to folded. At the highest surface coupling, the entropic penalty of folding vanishes, and a folding intermediate is strongly stabilized. In this intermediate state, the α-domain of lysozyme is disrupted, while the β-sheet remains fully structured. We rationalize the relative stability of the two domains on the basis of the residue contact order.     

Streaming potential and protein transmission ultrafiltration of single proteins and proteins in mixture: β-lactoglobulin and lysozyme

A study of single proteins, β-lactoglobulin and lysozyme of different sizes and electrical characteristic as a function of pH, ionic strength and nature of salt (NaCl or CaCl₂) allows to evaluate the filtration performances. Streaming potential measurements confirm that proteins contribute to the net charge of the system and that the protein tranfer through mineral membranes is governed by ionic and steric exclusion phenomena.This work shows the correlation between protein transmission and streaming potential values which takes into account steric and ionic exclusion. The ionic repulsion decreases the transmission. This one depends on membrane net charge characterised by streaming potential, which depends on the solution composition. This model, which does not take into account interactions between protein and membrane fouling leads to an overestimation of calculated transmission values when proteins are in a mixture. However, it allows a correct estimation of transmission variations versus the studied variables: pH and ionic strength.     

Theoretical studies on the interactions and interferences of HIV-1 glycoprotein gp120 and its coreceptor CCR5

The interaction between the HIV gp120 protein and coreceptor CCR5 or CXCR4 of the host cell is critical in mediating the HIV entry process. A model for the CCR5-gp120 complex has been developed. In the model, the N-terminus of CCR5 binds to three discontinuous domains of gp120, including the fourth conserved (C4) region, β19/β20 connecting loop, and V3 loop. The second extra-cellular loop (ECL2) of CCR5 also interacts with the crown part of the gp120 V3 loop. The bindings of the three CCR5 antagonists, maraviroc, aplaviroc, and vicriviroc, to the trans-membrane domain of CCR5 have been modeled. The bindings are found to affect the conformation of the ECL2 domain, which in turn drives the N-terminus of CCR5 to an altered state. Aplaviroc is more hydrophilic than maraviroc and vicriviroc, and its binding is more interfered by solvent, resulting in a quite different effect to the structure of CCR5 compared with those of the other two molecules. The above results are in accord with experimental observations and provide a structural basis for further design of CCR5 antagonists.     

Mechanisms of inter- and intramolecular communication in GPCRs and G proteins

This study represents the first attempt to couple, by computational experiments, the mechanisms of intramolecular and intermolecular communication concerning a guanidine nucleotide exchange factor (GEF), the thromboxane A2 receptor (TXA2R), and the cognate G protein (Gq) in its heterotrimeric GDP-bound state. Two-way pathways mediate the communication between the receptor-G protein interface and both the agonist binding site of the receptor and the nucleotide binding site of the G protein. The increase in solvent accessibility in the neighborhoods of the highly conserved E/DRY receptor motif, in response to agonist binding, is instrumental in favoring the penetration of the C-terminus of Gqalpha in between the cytosolic ends of H3, H5, and H6. The arginine of the E/DRY motif is predicted to be an important mediator of the intramolecular and intermolecular communication involving the TXA2R. The receptor-G protein interface is predicted to involve multiple regions from the receptor and the G protein alpha-subunit. However, receptor contacts with the C-terminus of the alpha5-helix seem to be the major players in the receptor-catalyzed motion of the alpha-helical domain with respect to the Ras-like domain and in the formation of a nucleotide exit route in between the alphaF-helix and beta6/alpha5 loop of Gqalpha. The inferences from this study are of wide interest, as they are expected to apply to the whole rhodopsin family, given also the considerable G protein promiscuity.     

Histidine Tautomerism Driving Human Islet Amyloid Polypeptide Aggregation in the Early Stages of Diabetes Mellitus Progression: Insight at the Atomistic Level

Early oligomerization of human islet amyloid polypeptide (hIAPP), which is accountable for β-cell death, has been implicated in the progression of type 2 diabetes mellitus. Some researches have shown the connection between hIAPP and Alzheimer's disease as well. However, the mechanism of peptide accumulation and associated cytotoxicity remains unclear. Due to the unique properties and significant role of histidine in protein sequences, here for the first time, the tautomeric effect of histidine at the early stages of amylin misfolding was investigated via molecular dynamics simulations. Considering Tau and Pi tautomeric forms of histidine (Tau and Pi tautomers are denoted as ϵ and δ, respectively), simulations were performed on two possible isomers of amylin. Our analysis revealed a higher probability of transient α-helix generation in the δ isomer in monomeric form. In dimeric forms, the δδ and δϵ conformations showed an elevated amount of α-helix and lower coil in comparison to the ϵϵ dimer. Due to the significant role of α-helix in membrane disruption and transition to β-sheet structure, these results may imply a noticeable contribution of the δ isomer and the δδ and δϵ dimers rather than ϵ and ϵϵ conformations in the early stages of diabetes initiation. Our results may aid in elucidating the hIAPP self-association process in the etiology of amyloidosis.     

Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane‐Spanning Protein

Mapping the interaction sites between membrane‐spanning proteins is a key challenge in structural biology. In this study a carbene‐footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine‐based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane‐spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein–protein interactions between the trimer subunits remained unlabelled, thus revealing their location.