Biomedical subjects

Isaiah T Arkin

Publications and source records attributed to Isaiah T Arkin.

At least 19 recordsLinked to original sources

PubMed2006

Structural disorder of the CD3zeta transmembrane domain studied with 2D IR spectroscopy and molecular dynamics simulations.

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528] we used 2D IR spectroscopy and 1-(13)C=(18)O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3zeta transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the alpha-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

CD3 Complex↗

PubMed2006

A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain.

In an attempt to understand what distinguishes severe acute respiratory syndrome (SARS) coronavirus (SCoV) from other members of the coronaviridae, we searched for elements that are unique to its proteins and not present in any other family member. We identified an insertion of two glycine residues, forming the GxxxG motif, in the SCoV spike protein transmembrane domain (TMD), which is not found in any other coronavirus. This surprising finding raises an "oligomerization riddle": the GxxxG motif is a known dimerization signal, while the SCoV spike protein is known to be trimeric. Using an in vivo assay, we found that the SCoV spike protein TMD is oligomeric and that this oligomerization is driven by the GxxxG motif. We also found that the GxxxG motif contributes toward the trimerization of the entire spike protein; in that, mutations in the GxxxG motif decrease trimerization of the full-length protein expressed in mammalian cells. Using molecular modeling, we show that the SCoV spike protein TMD adopts a distinct and unique structure as opposed to all other coronaviruses. In this unique structure, the glycine residues of the GxxxG motif are facing each other, enhancing helix-helix interactions by allowing for the close positioning of the helices. This unique orientation of the glycine residues also stabilizes the trimeric bundle during multi-nanosecond molecular dynamics simulation in a hydrated lipid bilayer. To the best of our knowledge, this is the first demonstration that the GxxxG motif can potentiate other oligomeric forms beside a dimer. Finally, according to recent studies, the stabilization of the trimeric bundle is linked to a higher fusion activity of the spike protein, and the possible influence of the GxxxG motif on this feature is discussed.

Amino Acid Motifs↗

PubMed2006

Isotope-edited IR spectroscopy for the study of membrane proteins.

Fourier transform infrared (FTIR) spectroscopy has long been a powerful tool for structural analysis of membrane proteins. However, because of difficulties in resolving contributions from individual residues, most of the derived measurements tend to yield average properties for the system under study. Isotope editing, through its ability to resolve individual vibrations, establishes FTIR as a method that is capable of yielding accurate structural data on individual sites in a protein.

Carbon Isotopes↗

PubMed2006

Picosecond dynamics of a membrane protein revealed by 2D IR.

Fast protein dynamics can be missed with techniques that have relatively slow observation times. Using 2D IR spectroscopy and isotope labeling, we have probed the rapid, picosecond dynamics of a membrane protein in its native environment. By measuring the homogeneous and inhomogeneous IR linewidths of 11 amide I modes (backbone carbonyl stretch), we have captured the structural distributions and dynamics of the CD3zeta protein along its transmembrane segment that are lost with slower time-scale techniques. We find that the homogeneous lifetimes and population relaxation times are the same for almost all of the residues. In contrast, the inhomogeneous linewidths vary significantly with the largest inhomogeneous distribution occurring for residues near the N terminus and the narrowest near the center. This behavior is highly consistent with a recently reported experimental model of the protein and water accessibility as observed by molecular dynamics simulations. The data support the proposed CD3zeta peptide structure, and the simulations point to the structural disorder of water and lipid head-groups as the main source of inhomogeneous broadening. Taken together, this rigorous analysis of the vibrational dynamics of a membrane peptide provides experimental insight into a time regime of motions that has so far been largely unexplored.

CD3 Complex↗

PubMed2005

Disorder influence on linear dichroism analyses of smectic phases.

Linear dichroism, the unequal absorption of parallel and perpendicular linear polarized light, is often used to determine the anisotropic ordering of rodlike polymers in a smectic phase, such as helices in a lipid bilayer. It is a measure of two properties of the sample: 1), orientation of the chromophore transition dipole moment (TDM) and 2), disorder. Since it is the orientation of the chromophore TDM that is needed for high resolution structural studies, it is imperative to either deconvolve sample disorder, or at a minimum, estimate its effect upon the calculated TDM orientation. Herein, a rigorous analysis of the effects of disorder is undertaken based on the recently developed Gaussian disorder model implemented in linear dichroism data. The calculation of both the rod tilt and rotational pitch angles as a function of the disorder and dichroism, yield the following conclusions: Disorders smaller than 5 degrees have a vanishingly small effect on the calculated polymer orientation, whereas values smaller than 10 degrees have a negligible effect on the calculated parameters. Disorders larger than 10 degrees have an appreciable effect on the calculated orientational parameters and as such must be estimated before any structural characterization. Finally the theory is tested on the HIV vpu transmembrane domain, employing experimental mosaicity measurements from x-ray reflectivity rocking scans and linear dichroism.

Biophysics↗

PubMed2005

Genetic algorithm-based optimization of hydrophobicity tables.

SUMMARY: The genomic abundance and pharmacological importance of membrane proteins have fueled efforts to identify them based solely on sequence information. Previous methods based on the physicochemical principle of a sliding window of hydrophobicity (hydropathy analysis) have been replaced by approaches based on hidden Markov models or neural networks which prevail due to their probabilistic orientation. In the current study, an optimization of the hydrophobicity tables used in hydropathy analysis is performed using a genetic algorithm. As such, the approach can be viewed as a synthesis between the physicochemically and statistically based methods. The resulting hydrophobicity tables lead to significant improvement in the prediction accuracy of hydropathy analysis. Furthermore, since hydropathy analysis is less dependent on the basis set of membrane proteins is used to hone the statistically based methods, as well as being faster, it may be valuable in the analysis of new genomes. Finally, the values obtained for each of the amino acids in the new hydrophobicity tables are discussed.

Algorithms↗

PubMed2005

A periodicity analysis of transmembrane helices.

Transmembrane helices and the helical bundles which they form are the major building blocks of membrane proteins. Since helices are characterized by a given periodicity, it is possible to search for patterns of traits which typify one side of the helix and not the other (e.g. amphipathic helices contain a polar and apolar sides). Using Fourier transformation we have analyzed solved membrane protein structures as well as sequences of membrane proteins from the Swiss-Prot database. The traits searched included aromaticity, volume and ionization. While a number of motifs were already recognized in the literature, many were not. One particular example involved helix VII of lactose permease which contains seven aromatic residues on six helical turns. Similarly six glycine residues in four consecutive helical turns were identified as forming a motif in the chloride channel. A tabulation of all the findings is presented as well as a possible rationalization of the function of the motif.

Algorithms↗

PubMed2005

How pH opens a H+ channel: the gating mechanism of influenza A M2.

The tetrameric M2 protein from influenza A is one of the simplest pH-gated H+ channels known, offering the potential of structurally characterizing its gating mechanism. Since the only ionizable groups in the pore are four histidines, we investigated the stability and dynamics of all six possible protonation states of the protein by using molecular dynamics. We show that while all channel protonation states are surprisingly stable, only systems with two or more charged histidines are appreciably conductive. The structural switch, from a uniprotonated to a biprotonated channel, causes an electrostatic repulsion between the charged histidines that pushes the helices apart. This results in the formation of a continuous water file that conducts protons via a H+ wire. pKa calculations place this transition at a pH of 5.6, in remarkable agreement with the experimental value. Since the conversion from uniprotonation to biprotonation occurs during endosome acidification, this explains how M2 is activated in vivo.

Histidine↗

PubMed2004

A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein.

The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E protein's pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

Amino Acid Sequence↗

PubMed2004

Site-specific vibrational dynamics of the CD3zeta membrane peptide using heterodyned two-dimensional infrared photon echo spectroscopy.

Heterodyned two-dimensional infrared (2D IR) spectroscopy has been used to study the amide I vibrational dynamics of a 27-residue peptide in lipid vesicles that encompasses the transmembrane domain of the T-cell receptor CD3zeta. Using 1-(13)C[Double Bond](18)O isotope labeling, the amide I mode of the 49-Leucine residue was spectroscopically isolated and the homogeneous and inhomogeneous linewidths of this mode were measured by fitting the 2D IR spectrum collected with a photon echo pulse sequence. The pure dephasing and inhomogeneous linewidths are 2 and 32 cm(-1), respectively. The population relaxation time of the amide I band was measured with a transient grating, and it contributes 9 cm(-1) to the linewidth. Comparison of the 49-Leucine amide I mode and the amide I band of the entire CD3zeta peptide reveals that the vibrational dynamics are not uniform along the length of the peptide. Possible origins for the large amount of inhomogeneity present at the 49-Leucine site are discussed.

CD3 Complex↗

PubMed2004

Experimental measurement of the strength of a C alpha-H...O bond in a lipid bilayer.

Due to the apolar nature of the lipid bilayer, the weak Calpha-H...O H-bond is thought to contribute significantly toward the stability of transmembrane helical bundles such as glycophorin A (GPA). Here for the first time we measured the strength of such a bond, using vibrational frequency shifts of a dimeric and nondimeric variants of GPA containing a Gly CD2 label. Although the resulting estimated bond strength of 0.88 kcal/mol is relatively weak, several such bonds could contribute significantly toward bundle stabilization.

Carbon↗

PubMed2004

A novel method of resistance for influenza against a channel-blocking antiviral drug.

Effective antivirals are few and far between, and as such, the appearance of resistance toward such treatments is an obvious medical concern. In this article, we analyze the mechanism by which influenza attains resistance toward amantadine, a blocker of the viral M2 H(+) channel. Binding analyses of amantadine to M2 peptides from different viral strains showed that the virus has developed two alternate routes to avoid blockage of its channel: (1) a conventional route, in which the channel no longer binds the blocker and, hence, the blocker cannot exert its inhibitory function; and (2) a novel mechanism, in which binding of the blocker is retained, yet the function of the protein is unaffected. Pore diameter profiles revealed the molecular mechanism by which the virus may attain this novel type of resistance: an increase in the size of the channel. Thus, despite the drug binding the channel, it may not be able to block the pore, since the channel diameter has increased. Our findings may have broad ramifications in the design of new antivirals, and of novel blockers against malfunctioning human channels implicated in disease.

Amantadine↗

PubMed2004

Modeling sample disorder in site-specific dichroism studies of uniaxial systems.

Site-specific infrared dichroism is an emerging method capable of proposing a model for the backbone structure of a transmembrane alpha-helix within a helical bundle. Dichroism measurements of single, isotopically enhanced vibrational modes (e.g., Amide I 13C=18O or Gly CD2 stretching modes) can yield precise orientational restraints for the monomer helix protomer that can be used as refinement constraints in model building of the entire helical bundle. Essential, however, for the interpretation of the dichroism measurements, is an accurate modeling of the sample disorder. In this study we derive an enhanced and more realistic modeling of the sample disorder based on a Gaussian distribution of the chromophore around a particular angle. The enhanced utility of the Gaussian model is exemplified by the comparative data analysis based on the aforementioned model to previously employed models.

Circular Dichroism↗

PubMed2004

Structural conservation in the major facilitator superfamily as revealed by comparative modeling.

The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published. Despite the low sequence similarity of the two proteins involved, lactose permease and glycerol-3-phosphate transporter, the reported structures are highly similar. This leads to the hypothesis that all members of the MFS share a similar structure, regardless of their low sequence identity. To test this hypothesis, we generated models of two other members of the MFS, the Tn10-encoded metal-tetracycline/H(+) antiporter (TetAB) and the rat vesicular monoamine transporter (rVMAT2). The models are based on the two MFS structures and on experimental data. The models for both proteins are in good agreement with the data available and support the notion of a shared fold for all MFS proteins.

Animals↗

PubMed2004

Distinct protein interfaces in transmembrane domains suggest an in vivo folding model.

Given the known high-resolution structures of alpha-helical transmembrane domains, we show that there are statistically distinct classes of transmembrane interfaces which relate to the folding and oligomerization of transmembrane domains. Distinct types of interfaces have been categorized and refer to those between: the same polypeptide chain, different polypeptide chains, helices that are sequential neighbors, and those that are nonsequential. These different interfaces may reflect different phases in the mechanism of transmembrane domain folding and are consistent with the current experimental evidence pertaining to the folding and oligomerization of transmembrane domains. The classes of helix-helix interfaces have been identified in terms of the numbers and different types of pairwise amino acid interactions. The specific measures used are interaction entropy, the information content of interacting partners compared to a random set of contacts, the amino acid composition of the classes and the abundances of specific amino acid pairs in close contact. Knowledge of the clear differences in the types of helix-helix contacts helps with the derivation of knowledge-based constraints which until now have focused on only the interiors of transmembrane domains as compared to the exterior. Taken together, an in vivo model for membrane protein folding is presented, which is distinct from the familiar two-stage model. The model takes into account the different interfaces of membrane helices defined herein, and the available data regarding folding in the translocation channel.

Databases, Protein↗

PubMed2003

Monte Carlo estimation of the number of possible protein folds: effects of sampling bias and folds distributions.

The estimation of the number of protein folds in nature is a matter of considerable interest. In this study, a Monte Carlo method employing the broken stick model is used to assign a given number of proteins into a given number of folds. Subsequently, random, integer, non-repeating numbers are generated in order to simulate the process of fold discovery. With this conceptual framework at hand, the effects of two factors upon the fold identification process were investigated: (1) the nature of folds distributions and (2) preferential sampling bias of previously identified folds. Depending on the type of distribution, dividing 100,000 proteins into 1,000 folds resulted in 10-30% of the folds having 10 proteins or less per fold, approximately 10% of the folds having 10-20 proteins per fold, 31-45% having 20-100 proteins per fold, and >30% of the folds having more than 100 proteins per fold. After randomly sampling one tenth of the proteins, 68-96% of the folds were identified. These percentages depend both on folds distribution and biased/non-biased sampling. Only upon increasing the sampling bias for previously identified folds to 1,000, did the model result in a reduction of the number of proteins identified by an order of magnitude (approximately 9%). Thus, assuming the structures of one tenth of the population of proteins in nature have been solved, the results of the Monte Carlo simulation are more consistent with recent lower estimates of the number of folds, <or=1,000. Any deviation from this estimate would reflect significant bias in the experimental sampling of protein structure, and/or substantially nonuniform folds distribution, manifested in a large number of single-fold proteins.

Models, Theoretical↗

PubMed2003

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner.

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.

Animals↗

PubMed2003

Site-specific dichroism analysis utilizing transmission FTIR.

Infrared spectroscopy has long been used to examine the average secondary structure and orientation of membrane proteins. With the recent utilization of site-specific isotope labeling (e.g., peptidic 1-(13)C = (18)O) it is now possible to examine localized properties, rather than global averages. The technique of site-specific infrared dichroism (SSID) capitalized on this fact, and derives site-specific orientational restraints for the labeled amino acids. These restraints can then be used to solve the backbone structure of simple alpha-helical bundles, emphasizing the capabilities of this approach. So far SSID has been carried out in attenuated total internal reflection optical mode, with all of the respective caveats of attenuated total internal reflection. In this report we extend SSID through the use of transmission infrared spectroscopy tilt series. We develop the corresponding theory and demonstrate that accurate site-specific orientational restraints can be derived from a simple transmission experiment.

Algorithms↗