Helical Sequences Research Articles

Spatial programming of defect distributions to enhance material failure characteristics

Defects play a major role in determining the mechanical properties of materials. Examples of this span from dislocations, grain boundaries, and precipitates in metallurgy to voids and imperfect interfaces in natural composites. These can enable complex failure modes and balance competing mechanical properties. With the increasing adoption of additive manufacturing in both research and industry, there are unprecedented opportunities for controlling the internal composition and structure of a material system. However, the very nature of forming a material in increments produces a set of characteristic defects (e.g., void formation due to incomplete merger of new material), which results in local heterogeneity and anisotropy. Rather than seeking to prevent these characteristic defects, we utilize them to improve the damage resistance of printed structures by systematically controlling their distribution within a single material. Inspired by the well-known Bouligand structure found in tough natural materials, we use a helical build sequence in which each layer is added at a defined pitch angle relative to the previous layer. The resulting helical defect distribution can guide the crack tip during fracture and enhance damage resistance. Microstructural evidence from crack surface images and X-ray microtomography reveals intricate mixing of twisting and branching cracks during failure, and is explained via an analytical model. Since these improvements are obtained solely from the defect distribution within a single material, the findings of this work could improve the failure characteristics of a broad range of printed materials (metals, ceramics, polymers, and composites).

Functional Coiled-Coil-like Assembly by Knob-into-Hole Packing of Single Heptad Repeat.

Coiled-coil peptides represent the principal building blocks for structure-based design of bionanomaterials. The sequence-structure relationship and precise nanoscale ordering of the coiled-coil helices originate from the knob-into-hole (KIH) packing of side chains. The helical interface stabilized by the KIH interaction is known to have chain lengths ranging from 30 to 1000 residues. Yet the shortest peptide required for oligomerization through KIH assembly is still unknown. Here, we report that through atomic resolution a minimal seven-residue amphipathic helix forms a different type of KIH motif, termed "supramolecular KIH packing", which confers an exceptional stability to the helical dimers. Significantly, at a low pH, the peptide self-assembles into nanofibers with coiled-coil architecture resembling the natural fibrous proteins. Furthermore, hierarchical ordering of the nanofibers affords lyotropic liquid crystals composed of a shortest natural helical sequence. Thus, this study expands the sequence space for a coiled-coil folding manifold and provides another paradigm for designer nanomaterials from minimal helical sequences.

Synthesis of a Novel AB Block Copolyaceylene Consisting of a Dynamic Cis-transoidal Racemic Helical Sequence and a Static Cis-cisoidal One-handed Helical Sequence

We have successfully synthesized a novel chiral AB block copolyacetylene consisting of a dynamic cis-transoidal racemic helical sequence and a static cis-cisoidal one-handed helical sequence by usi...

Improving the diagnosis of peripheral arterial disease in below-the-knee arteries by adding time-resolved CT scan series to conventional run-off CT angiography. First experience with a 256-slice CT scanner

PurposeRun-off Computed Tomography Angiography (run-off CTA) of the lower extremities has become the method of choice for the diagnostic imaging of patients suffering from peripheral arterial disease (PAD). However, it remains a challenging radiological examination with a considerable risk of non-diagnostic image quality for the assessment of below-the-knee arteries. In this study, we investigate the diagnostic benefit of adding time-resolved CT scan series to the standard run-off CTA by performing repeated axial acquisitions over the calves of the patient during a second bolus of iodinated contrast injection. Materials and MethodsThis prospective study included 20 patients (9 male, 11 female; mean age 66.1 ± 14.9 years) who received a standard run-off CTA and an additional time-resolved CT scan series after a 10 min delay. The time-resolved series consisted of 18 repeated axial acquisitions over the calves directly below the knee with a 2 s interphase delay. For both series, two observers independently assessed the anterior tibial, posterior tibial and peroneal arteries of both legs for following criteria: arterial enhancement, presence and degree of stenosis, the confidence of grading, degree of stenosis and venous overlay. Quantitative assessment of arterial enhancement was performed by measuring the mean CT values (HU) in all arteries. Radiation exposure was quantified by the effective dose. ResultsA total of 118 arteries were assessed. The observer study showed that the additional time-resolved series improved both arterial enhancement (64% considered optimal enhanced versus 44%) and diagnostic confidence (59% considered as certain versus 33%) for the assessment of arterial stenosis (all p < 0.05). Venous overlay reduced from 15% to 6%. In all three arteries, the measured contrast enhancement by CT values (HU) was considerably higher (average 48%, p < 0.05) with the time-resolved series. The time-resolved series had an effect on stenosis classification (p = 0.03): a higher number of arteries were graded as having a non-significant stenosis (78.8% versus 71.2%). The interobserver variability in stenosis classification improved from κ = 0.39 to κ = 0.61. The mean effective dose was 5.1 ± 1.3 mSv for the run-off CTA and 0.2 ± 0.07 mSv for the time-resolved series. Per patient, a total volume of 140 mL contrast agent was injected. ConclusionA dynamic CT scan protocol with repeated axial series can be added to a standard helical run-off CTA sequence for the lower extremities within the same CT examination, and it increases image quality and diagnostic confidence for the assessment of presence and degree of arterial stenosis in below-the-knee arteries.

Assembly of Multicomponent Protein Filaments Using Engineered Subunit Interfaces.

Exploiting the ability of proteins to self-assemble into architectural templates may provide novel routes for the positioning of functional molecules in nanotechnology. Here we report the engineering of multicomponent protein templates composed of distinct monomers that assemble in repeating orders into a dynamic functional structure. This was achieved by redesigning the protein-protein interfaces of a molecular chaperone with helical sequences to create unique subunits that assemble through orthogonal coiled-coils into filaments up to several hundred nanometers in length. Subsequently, it was demonstrated that functional proteins could be fused to the subunits to achieve ordered alignment along filaments. Importantly, the multicomponent filaments had molecular chaperone activity and could prevent other proteins from thermal-induced aggregation, a potentially useful property for the scaffolding of enzymes. The design in this work is presented as proof-of-concept for the creation of modular templates that could potentially be used to position functional molecules, stabilize other proteins such as enzymes, and enable controlled assembly of nanostructures with unique topologies.

Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling.

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-Rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag ('dC10*') was then demonstrated in the fluorogenic intracellular labelling of histone H2B in living HeLa cells.

NMR Investigation about Heterogeneous Structure and Dynamics of Recombinant Spider Silk in the Dry and Hydrated States

Spider silks continue to attract researchers because of their excellent mechanical properties and supercontraction behavior. In this paper, the structure and dynamics of recombinant spider silk protein (RSP) were characterized using 13C CP/MAS, 13C DD/MAS, and 13C refocused-INEPT NMR spectroscopies in the dry and hydrated states. The fractions of several structures of RSP with helical, random coil, and β-sheet polyalanine sequences were determined from the CP/MAS NMR spectra in the dry state. The CP/MAS NMR spectra changed to very simple one with dominant β-sheet Ala peaks by hydration due to a significant loss in CP signals of the other mobile carbons. On the contrary, only sharp mobile peaks, and both mobile and immobile peaks could be observed in the refocused-INEPT and DD/MAS NMR spectra, respectively. The cis/trans proportion of the Gly–Pro bond was also determined. Our measurements provide new insight into understanding the supercontraction phenomenon of spider silks.

Revisiting the Helical Cooperativity of Synthetic Polypeptides in Solution.

Using synthetic polypeptides as a model system, the theories of helix-coil transition were developed into one of the most beautiful and fruitful subjects in macromolecular science. The classic models proposed by Schellman and Zimm-Bragg more than 50 years ago, differ in the assumption on whether the configuration of multiple helical sequences separated by random coil sections is allowed in a longer polypeptide chain. Zimm also calculated the critical chain lengths that facilitate such interrupted helices in different solvent conditions. The experimental validation of Zimm's prediction, however, was not carefully examined at that time. Herein, we synthesize a series of homopolypeptide samples with different lengths, to systematically examine their helix-coil transition and folding cooperativity in solution. We find that for longer chains, polypeptides do exist as interrupted helices with scattered coil sections even in helicogenic solvent conditions, as predicted in the Zimm-Bragg model. The critical chain lengths that facilitate such interrupted helices, however, are substantially smaller than Zimm's original estimation. The inaccuracy is in part due to an approximation that Zimm made in simplifying the calculation. But more importantly, we find there exist intramolecular interactions between different structural segments in the longer polypeptides, which are not considered in the classic helix-coil theories. As such, even the Zimm-Bragg model in its exact form cannot fully describe the transition behavior and folding cooperativity of longer polypeptides. The results suggest that long "all-helix" chains may be much less prevalent in solution than previously imagined, and a revised theory is required to accurately account for the helix-coil transition of the longer chains with potential "non-local" intramolecular interactions.

Abstract 3613: Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor

Abstract Approximately 70% of breast cancer cells express estrogen receptor alpha (ERα), and depend on estrogen (E2) for proliferative and metastatic activity. The current endocrine therapies for breast cancer are mainly based on targeting ERα signaling using selective ERα modulators, ERα downregulators, and aromatase inhibitor. However, up to 50% of patients with ERα-positive tumors either initially do not respond or become resistant to these drugs. The precise molecular mechanisms for the endocrine resistance contributes to be an active area of research. Therefore, identifying the factors and pathways responsible for resistance and defining ways to overcome it lead to develop novel molecular-target therapies to endocrine resistance. Recent findings support that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signaling modulation in these cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids) derive from a helical BIG3 sequence, a dominant-negative peptide inhibitor leads to the significant anti-tumor effect. However, duration of its effect is very short for clinical use. Here, we report the development of the chemically modified ERAP using stapling methods (stapled ERAP) to improve duration of its antitumor effects. The stapled ERAP specifically inhibited the BIG3-PHB2 interaction, thereby exhibiting the long-lasting suppressive activity and their intracellular localization without membrane-permeable polyarginine sequence supposedly through the formation of stable α-helix structure induced by the stapling. Importantly, a combination of stapled ERAP and tamoxifen caused a synergistic inhibitory effect in breast cancer cell growth. Tumor bearing mice treated with every 7 days with stapled ERAP treatment effectively prevented the BIG3-PHB2 interaction as well as daily treatment, leading to the complete regression of E2-dependent tumor in vivo. Our findings suggest that the stapled ERAP may be a promising anti-tumor drug to suppress the growth of luminal-type breast cancer in clinical use. Citation Format: Toyomasa Katagiri, Tetsuro Yoshimaru. Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2017-3613

Downsizing Proto-oncogene cFos to Short Helix-Constrained Peptides That Bind Jun.

The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT48-57) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.

RNAHelix: computational modeling of nucleic acid structures with Watson-Crick and non-canonical base pairs.

Comprehensive analyses of structural features of non-canonical base pairs within a nucleic acid double helix are limited by the availability of a small number of three dimensional structures. Therefore, a procedure for model building of double helices containing any given nucleotide sequence and base pairing information, either canonical or non-canonical, is seriously needed. Here we describe a program RNAHelix, which is an updated version of our widely used software, NUCGEN. The program can regenerate duplexes using the dinucleotide step and base pair orientation parameters for a given double helical DNA or RNA sequence with defined Watson-Crick or non-Watson-Crick base pairs. The original structure and the corresponding regenerated structure of double helices were found to be very close, as indicated by the small RMSD values between positions of the corresponding atoms. Structures of several usual and unusual double helices have been regenerated and compared with their original structures in terms of base pair RMSD, torsion angles and electrostatic potentials and very high agreements have been noted. RNAHelix can also be used to generate a structure with a sequence completely different from an experimentally determined one or to introduce single to multiple mutation, but with the same set of parameters and hence can also be an important tool in homology modeling and study of mutation induced structural changes.

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens.

In-situ investigation on the structural evolution of mesomorphic isotactic polypropylene in a continuous heating process

Abstract The microstructural changes of mesomorphic iPP from mesophase to α transition at a molecular level in a continuous heating process have been studied by in-situ Fourier-transform infrared (FT-IR) spectroscopy, in-situ X-ray scattering using synchrotron radiation, differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). Microstructural change corresponding to helical conformation variation in the temperature range between 20 and 60 °C was detected by IR spectroscopy, which may originate from the glass transition of rigid amorphous fraction (RAF). The helical sequence with 12 monomer units is found to exist in RAF. The contents of helical sequences with different number of monomers exhibit different variation trends in the course of meso-α transition and the following process of partial melting and perfection of α crystal. A plausible mechanism was proposed that RAF experiences glass transition firstly at low temperature, and then serve as α nuclei to trigger the meso-α transition at higher temperature. This work provides a new insight into the mechanism of microstructural evolution of the meso-α transformation of iPP.

Ionization Properties of Histidine Residues in the Lipid Bilayer Membrane Environment

We address the critically important ionization properties of histidine side chains of membrane proteins, when exposed directly to lipid acyl chains within lipid bilayer membranes. The problem is important for addressing general principles that may underlie membrane protein function. To this end, we have employed a favorable host peptide framework provided by GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-amide). We inserted His residues into position 12 or 14 of GWALP23 (replacing either Leu(12) or Leu(14)) and incorporated specific [(2)H]Ala labels within the helical core sequence. Solid-state (2)H NMR spectra report the folding and orientation of the core sequence, revealing marked differences in the histidine-containing transmembrane helix behavior between acidic and neutral pH conditions. At neutral pH, the GWALP23-H12 and GWALP23-H14 helices exhibit well defined tilted transmembrane orientations in dioleoylphosphatidylcholine (DOPC)and dilauroylphosphatidylcholine (DLPC) bilayer membranes. Under acidic conditions, when His(12) is protonated and charged, the GWALP23-H12 helix exhibits a major population that moves to the DOPC bilayer surface and a minor population that occupies multiple transmembrane states. The response to protonation of His(14) is an increase in helix tilt, but GWALP23-H14 remains in a transmembrane orientation. The results suggest pKa values of less than 3 for His(12) and about 3-5 for His(14) in DOPC membranes. In the thinner DLPC bilayers, with increased water access, the helices are less responsive to changes in pH. The combined results enable us to compare the ionization properties of lipid-exposed His, Lys, and Arg side chains in lipid bilayer membranes.

Transmembrane Substrate Unfolding in Intramembrane Proteolysis

In recent years impressive progress has been made in our understanding of intramembrane cleaving proteases (iCliPs), enzymes that carry out proteolytic reactions within cell membranes, and their substrates, but basic biochemical questions such as what determines a transmembrane helix to be a substrate and how cleavage site is dictated remain unresolved. Much onus as to why we sill do not understand substrate specificity in these enzymes falls to the difficulties in monitoring enzyme-substrate interactions in a lipid or membrane-mimetic environment. Here we will present deep-UV resonance Raman spectroscopy data describing discrete structural characteristics of substrates prior to and during interactions with iCLiPS indicating structural flexibility in both environments is an intrinsic property of cleavable substrates. Furthermore, we also will present evidence that mutagenesis of substrates that abolishes intramembrane proteolysis do not significantly influence binding and dissociation constants of enzyme and substrate, while they do convey distinct changes to the intrinsic structural flexibility of the substrate. The mutations also alter the extent to which the enzyme influences the substrate structure during binding. Overall these results imply a synergy between a sequence complementarity between enzyme and substrate, which requires adequate structural flexibility of the substrate transmembrane helical sequence in order to expose the peptide bond for cleavage.

Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs.

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.

Environmental polarity induces conformational transitions in a helical peptide sequence from bacteriophage T4 lysozyme and its tandem duplicate: a molecular dynamics simulation study.

Our recent molecular dynamics (MD) simulation of an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme demonstrated a solvent induced α→β transition in a loosely held duplicate helical region, while α-helical conformation in the parent region was relatively stabilized by its tertiary interactions with the neighboring residues. The solution NMR of the parent helical sequence, sans its protein context, showed no inherent tendency to adopt a particular secondary structure in pure water but showed α-helical propensity in TFE/water and SDS micelles. In this study we investigate the conformational preference of the 'parent' and 'duplicate' sequences, sans the protein context, in pure water and an apolar TFE/water solution. Apolar TFE/water solution is a model for non-polar protein context. We performed MD simulations of the two peptides, in explicit water and 80% (v/v) TFE/water, using GROMOS 53a6 force field, at 300 K and 1 bar (under NPT conditions). We show that in TFE/water mixture, salt bridges are stabilized by apolar TFE molecules and main chain-main chain hydrogen bonds promote the α-helical conformation, particularly in the duplicate peptide. Solvent exposure, in pure water, resulted in an α→β transition to form a triple stranded β-sheet structure in the 'duplicate' sequence, with a rare psi-loop topology, while a mixture of turn/bend conformations were adopted by the 'parent' sequence. Thus the differences in conformational preference of the parent and duplicate sequence sans protein context, in pure water and TFE/water, implicate the importance of the environment polarity in dictating the peptide conformation. Mechanism of folding of the observed psi-loop in the duplicate sequence gives insights into folding of this rare β-sheet topology.

Multiple structural states exist throughout the helical nucleation sequence of the intrinsically disordered protein stathmin, as reported by electron paramagnetic resonance spectroscopy.

The intrinsically disordered protein (IDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.

An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity.

The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from aCD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying atruncated Env (CT84). To determine whether aspecific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared aseries of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred aswitch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.