Degree Of Secondary Structure Research Articles

Investigation of Sequence-Penetration Relationships of Antisense Oligonucleotides.

A major limitation for the development of more effective oligonucleotide therapeutics has been a lack of understanding of their penetration into the cytosol. While prior work has shown how backbone modifications affect cytosolic penetration, it is unclear how cytosolic penetration is affected by other features including base composition, base sequence, length, and degree of secondary structure. We have applied the chloroalkane penetration assay, which exclusively reports on material that reaches the cytosol, to investigate the effects of these characteristics on the cytosolic uptake of druglike oligonucleotides. We found that base composition and base sequence had moderate effects, while length did not correlate directly with the degree of cytosolic penetration. Investigating further, we found that the degree of secondary structure had the largest and most predictable correlations with cytosolic penetration. These methods and observations add a layer of design for maximizing the efficacy of new oligonucleotide therapeutics.

Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

Controlling Structure and Dimensions of a Disordered Protein via Mutations.

The dimensions of intrinsically disordered proteins (IDPs) are sensitive to small energetic-entropic differences between intramolecular and protein–solvent interactions. This is commonly observed on modulating solvent composition and temperature. However, the inherently heterogeneous conformational landscape of IDPs is also expected to be influenced by mutations that can (de)stabilize pockets of local and even global structure, native and non-native, and hence the average dimensions. Here, we show experimental evidence for the remarkably tunable landscape of IDPs by employing the DNA-binding domain of CytR, a high-sequence-complexity IDP, as a model system. CytR exhibits a range of structure and compactness upon introducing specific mutations that modulate microscopic terms, including main-chain entropy, hydrophobicity, and electrostatics. The degree of secondary structure, as monitored by far-UV circular dichroism (CD), is strongly correlated to average ensemble dimensions for 14 different mutants of CytR and is consistent with the Uversky–Fink relation. Our experiments highlight how average ensemble dimensions can be controlled via mutations even in the disordered regime, the prevalence of non-native interactions and provide testable controls for molecular simulations.

Hydrophobicity, rather than secondary structure, is essential for the SRP dependent targeting of GPR35 to the ER membrane

The folding and targeting of hydrophobic transmembrane domains poses a major challenge to the cell. Several membrane proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. However, there is little information on one of the largest classes of eukaryotic membrane proteins; the G protein-coupled receptors (GPCRs). In this study we show that the signal anchor domain of GPR35 remains in an extended conformation whilst exiting the ribosome tunnel, the polypeptide chain then forms interactions with components of the SRP targeting pathway, and the Sec61 translocon, resulting in a compacted conformation prior to integration into the ER membrane. We conclude that transmembrane structure is most likely adopted after the domain leaves the ribosome tunnel and that the interaction of the signal anchor with SRP is dependent on the native levels of hydrophobicity within the first transmembrane domain. Therefore, we propose a mechanism by which the first transmembrane domains of multi-spanning membrane proteins adopt compacted structures following SRP targeting but before insertion into the ER membrane.

An Evolutionary Algorithm for the Design of Different Degrees of Secondary Structure in Intrinsically Disordered Proteins (IDPs)

In recent experimental studies of coupled folding and binding of IDPs investigators have adapted methods from the protein folding literature whereby point mutations are used to identify the degree to which different regions are pre-folded prior to the rate limiting step in binding and folding. These approaches assume a one-to-one correspondence between coarse-grained readouts of structure such as alpha helicity and the degree of foldedness. Our previous work demonstrated a lack of correlation between alpha-helicity and the degree of disorder within unbound IDPs. Therefore, an important first step toward mutagenesis based approaches for dissecting the mechanisms of coupled folding and binding is the design of sequences that (a) have a target alpha helicity (b) have a prescribed degree of disorder and (c) retain the residues corresponding to the binding interface. We report results from an evolution algorithm (EA) that designs sequences to meet the specified criteria. We coupled this sequence design algorithm to atomistic simulations based on the ABSINTH model to quantify residue-specific helicities for each sequence. We demonstrate our approach using the sequence of PUMA, which folds upon binding to its target protein MCL-1. In the unbound ensemble, PUMA forms two uncorrelated helical segments that partition the sequence into two halves. In the bound complex, PUMA forms a single long helix. Our design strategy successfully generates sequences that span the spectrum of conformational options for the unbound PUMA that includes sequences with uncorrelated helical segments N- and C-terminal and sequences with a single helix that spans the entire sequence length. Our approach is well suited to the design of sequences for use in experiments geared toward dissecting the mechanisms of coupled folding and binding of IDPs.

Trifluoroethanol-Induced Changes in Activity and Conformation of Manganese-Containing Superoxide Dismutase

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, -9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.

Towards Al3+-Induced Manganese-Containing Superoxide Dismutase Inactivation and Conformational Changes: An Integrating Study with Docking Simulations

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in skins exposed to oxygen. We studied the inhibitory effects of Al3+ on the activity and conformation of manganese-containing SOD (Mn-SOD). Mn-SOD was significantly inactivated by Al3+ in a dose-dependent manner. The kinetic studies showed that Al3+ inactivated Mn-SOD follows the first-order reaction. Al3+ increased the degree of secondary structure of Mn-SOD and also disrupted the tertiary structure of Mn-SOD, which directly resulted in enzyme inactivation. We further simulated the docking between Mn-SOD and Al3+ (binding energy for Dock 6.3: −14.07 kcal/mol) and suggested that ASP152 and GLU157 residues were predicted to interact with Al3+, which are not located in the Mn-contained active site. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of Al3+ and allow us to describe a ligand binding via inhibition kinetics combined with the computational prediction.

Abstract 3156: eIF4E in human breast cancer and its role in regulating translation of splice variants of breast cancer genes

Abstract In eukaryotes, most of mRNAs are translated by the scanning mechanism. The trimeric complex eIF4F (consisting of eukaryotic initiation factor eIF4E, eIF4G and eIF4A) binds to ribosomes and the 5′end of mRNA, unwinding the secondary structures for scanning. eIF4E is the least abundant initiation factor, and may therefore determine the rate of translation. Overexpression of eIF4E can cause malignant transformation of immortalized human cells. Anti-sense oligonucleotides of eIF4E partially reversed the malignant phenotypes, suggesting that overexpression of eIF4E is the direct cause of transformation. Two- to threefold higher eIF4E levels have been detected in breast cancer using immunohistochemistry. Aabout 10% of cellular mRNAs contain atypically long 5’UTR and many of these encode proto-oncogenes and growth factors. It is believed that these long 5’UTR sequences tend to form stable secondary structure, requiring higher levels or activities of eIF4F for their translation. Over 60% of human genes are alternatively spliced. These may produce variants with different degrees of secondary structure at their 5’-UTR. We hypothesized that the elevated eIF4E level in breast cancer cells allows more effective translation of breast cancer gene variants with extensive 5’UTR secondary structure. These changes in variants profile may be important for breast cancer development and progression. We first analyzed the expression levels of eIF4E in human breast cancer and non-tumor biopsies in tissue microarray. Our preliminary results showed that there was significantly higher eIF4E expression in tumor versus non-tumor and eIF4E expression was positively correlated to estrogen receptor (ER) expression (OR=4.4, p=0.006). Although eIF4E expression levels were independent of tumor grade and size, it was strongly associated with overall clinical ER and progesterone receptor status (OR=6.3, p=0.003 and OR=6.5, p=0.002). The differential translation of breast cancer gene splice variants in breast cancer cell lines is being analyzed and their correlation with the extent of secondary structure of their 5’UTRs will be determined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3156.

Fesselin is a Natively Unfolded Protein

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg1 (TOAC0-BK) or replacing Pro3 (TOAC3-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism (CD) approach was used to examine the conformational properties of the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC3-BK's EPR spectral lineshapes are broader than those of TOAC0-BK, indicating a more restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC0-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidly interconverting forms), while TOAC3-BK's spectra correspond to a more ordered structure. SDS binding induces drastic changes in BK and TOAC0-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. In contrast. TOAC3-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC3-BK to acquire the correct receptor-bound conformation leading to loss of biological activity, On the other hand, the greater flexibility of TOAC0-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.

The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure.

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability.

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.

Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains

Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements.

Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular-dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30-51, 14-38) to complete refolding by forming directly the 5-55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest. 1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three-dimensional conformations. 2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure. 3. The degree of secondary structure already present in intermediates (30-51) seems to determine the pathway of refolding preferred by the corresponding protein.

Probing the RNA structure within the yeast 5 S RNA.L1a protein complex by fluorescence and enzymatic digestion.

Conformational states of the ribosomal 5 S RNA molecule and its associated protein L1a in the yeast RNA-protein (RNP) complex were determined using controlled RNase T1 digestion in conjunction with fluorescence probes, ethidium bromide and bisanilinonaphthalenesulfonic acid. Fluorescence measurements indicated that the RNA molecule in the RNP complex appeared to exhibit a slightly lower degree of secondary structure than that in the free form. Controlled digestion of the intact RNP complex with RNase T1 resulted in an initial increase in ethidium fluorescence followed by a gradual decrease. In free RNA, a similar profile, except that a larger increase in ethidium fluorescence at the initial stage of digestion, was observed. During digestion of the RNP complex, increases in bisanilinonaphthalenesulfonic acid fluorescence and in light scattering were observed. These findings implied that as regions of the 5 S RNA molecule were perturbed, hydrophobic regions in the protein became exposed. Polyacrylamide gel analysis of the digestion products revealed a temporal appearance of discrete RNA fragments. Sequence analysis of these fragments generated information about the structural arrangement of the RNA molecule within the RNP complex. Results from the present investigation indicate that interactions between the 5 S RNA and protein L1a can stabilize functionally relevant conformations of the components that are individually labile. Properties of the separated components also suggest that special conditions, such as those suggested by Steitz et al. (Steitz, J. A., Berg, C., Hendrick, J. P., LaBranche-Chabot, H., Metspalu, A., Rinke, J., and Yario, T. (1988) J. Cell Biol. 106, 545-556) may be involved for these components to associate during ribosomal assembly.

Properties of signal-sequence peptides at an air-water interface.

The surface behaviour of three signal-sequence polypeptides (the pretrypsinogen 2 signal sequence, a synthetic consensus signal sequence and the putative signal sequence of ovalbumin) were studied at an air-water interface. It was found that the surface stabilities of the spread polypeptide films were higher than those of polypeptides and proteins previously investigated (including melittin and membrane proteins), and that the signal peptides had a much lower affinity for the interface than had other peptides and proteins. The observed molecular areas of the signal-sequence peptides indicated that the molecules have a considerable degree of secondary structure at the surface interface.

Properties of smooth muscle vinculin.

Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Two-dimensional gel electrophoretic analysis of crude muscle fractions demonstrated that: 1) much of the approximately 130,000-dalton protein present in smooth muscle did not co-isoelectrically focus with the purified 130,000-dalton vinculin and 2) the purified vinculin consisted of three major, closely spaced isoelectric variants that were present only in small amounts in the original smooth muscle sample. Purified vinculin sedimented as a single peak with a sedimentation coefficient S0 20,w of 5.9. Circular dichroism spectra of purified vinculin indicated a considerable degree of secondary structure, with an alpha-helical content of approximately 50% as measured at 208 nm. The ultraviolet absorption spectrum of vinculin gave a measured E1%(278) of 4.64. Digestion of vinculin, much of which is located at the cytoplasmic surface of the cell membrane, with Ca2+-activated neutral protease purified from skeletal muscle yielded major fragments with molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for decreasing the low shear viscosity of actin was removed and found in a crude fraction isolated by CM-cellulose chromatography. The purified vinculin had a small, but positive effect on the MgCl2-induced polymerization of actin as measured by low shear viscometry.

Use of the neutron diffraction--H/D exchange technique to determine the conformational dynamics of trypsin.

A number of structural and chemical investigations (1–6) have shown that, even though protein molecules possess a high degree of secondary structure, they have numerous component segments which exhibit substantial fluctuations from their native conformations. A majority of these fluctuations represent a localized denaturation of small portions of the structure and result from the natural equilibrium between protein-protein and protein-solvent interactions. Although most of these fluctuations probably have little direct effect on the regions responsible for functional activity of the protein, a growing body of evidence indicates that conformational change is an important ingredient in a significant number of biological processes (7–10).

Capped mRNAs with Reduced Secondary Structure Can Function in Extracts from Poliovirus-Infected Cells

Extracts from poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, we demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosaic virus 4 RNA, which is most probably devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.