Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

“Setting paint” analogy for the hydrophobic self‐association of tropoelastin into elastin‐like hydrogel

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37°C. Reversion to a clear solution took place if the temperature was lowered to below 20°C within less than 2 h, but not if 37°C was maintained for several hours. The precipitated elastin‐like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37°C, but at 4°C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4°C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature‐controlled light microscopy analysis, ¹H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV‐absorption‐based concentration measurements. The self‐association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion‐like system in which the spherules coalesce in a manner like a water‐based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 321–330, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Association of bovine β‐casein protein variant I with milk production and milk protein composition

The aim of this study was to detect new polymorphisms in the bovine β‐casein (β‐CN) gene and to evaluate association of (new) β‐CN protein variants with milk production traits and milk protein composition. Screening of the β‐CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of β‐CN protein variant I in the Dutch HF population. Studies of association of β‐CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of β‐CN protein variants with milk composition was studied in 1857 first‐lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. β‐CN protein variant I was significantly associated with protein percentage and protein yield, and with α_s1‐casein (α_s1‐CN), α_s2‐casein (α_s2‐CN), κ‐casein (κ‐CN), α‐lactalbumin (α‐LA), β‐lactoglobulin (β‐LG), casein index and casein yield. Inferring β‐κ‐CN haplotypes showed that β‐CN protein variant I occurred only with κ‐CN variant B. Consequently, associations of β‐κ‐CN haplotype IB with protein percentage, κ‐CN, α‐LA, β‐LG and casein index are likely resulting from associations of κ‐CN protein variant B, while associations of β‐κ‐CN haplotype IB with α_s1‐CN and α_s2‐CN seem to be resulting from associations of β‐CN variant I.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

Kinetics of the self‐aggregation and film formation of poly‐L‐proline at high temperatures explored by circular dichroism spectroscopy

Poly‐L ‐proline has been used as a model system for various purposes over a period of more than 60 years. Its relevance among the protein/peptide community stems from its use as a reference system for determining the conformational distributions of unfolded peptides and proteins, its use as a molecular ruler, and from the pivotal role of proline residues in conformational transitions and protein–protein interactions. While several studies indicate that polyproline can aggregate and precipitate in aqueous solution, a systematic study of the aggregation process is still outstanding. We found, by means of UV‐circular dichroism and IR measurements, that polyproline is predominately monomeric at room temperature at millimolar concentrations. Upon heating, the polypeptide stays in its monomeric state until the temperature reaches a threshold of ca. 60°C. At higher temperatures, the peptide aggregates as a film on the inside surface of the employed cuvette. The process proceeds on a time scale of 10³ s and can best be described by a bi‐exponential relaxation function. The respective CD and IR spectra are qualitatively different from the canonical spectra of polyproline in aqueous solution, and are indicative of a highly packed state. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 451–457, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Real‐time detection of single‐living pancreatic β‐cell by laser tweezers Raman spectroscopy: High glucose stimulation

Glucose acts as a β‐cell stimulus factor and leads to cellular responses that involve a large amount of biomolecule formation, relocation, and transformation. We hypothesize that information about these changes can be obtained in real‐time by laser tweezers Raman spectroscopy. To test this hypothesis, repeated measurements designs in accordance with the application of Raman spectroscopy detection were used in the current experiment. Single rat β‐cells were measured by Raman spectroscopy in 2.8 mmol/l glucose culture medium as a basal condition. After stimulation with high glucose (20 mmol/l), the same cells were measured continuously. Each cell was monitored over a total time span of 25 min, in 5 min intervals. During this period of time, cells were maintained at an appropriate temperature controlled by an automatic heater, to provide near‐physiological conditions. It was found that some significant spectral changes induced by glucose were taking place during the stimulation time course. The most noticeable changes were the increase of spectral intensity at the 1002, 1085, 1445, and 1655 cm^?1 peaks, mainly corresponding to protein and lipid. We speculate that these changes might have to do with β‐cell protein and lipid synthesis. Using laser tweezers Raman spectroscopy in combination with glucose stimulation, optical spectral information from rat β‐cells was received and analyzed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 587–594, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

β‐Sheet aggregation of kisspeptin‐10 is stimulated by heparin but inhibited by amphiphiles

The murine 10‐residue neurohormone kisspeptin (YNWNSFGLRY) is an important regulator of reproductive behavior and gonadotrophin secretion. It is known to form a random coil in solution, but undergoes a structural change in the presence of membranes although the nature of this change is not fully determined. The peptide's conformational versatility raises the question whether it is also able to form ordered aggregates under physiological conditions, which might be relevant as a storage mechanism. Here we show that heparin induces kisspeptin to form β‐sheet rich amyloid aggregates both at neutral (pH 7.0) and slightly acidic (pH 5.2) conditions. Addition of heparin leads to aggregation after a certain lag phase, irrespective of the time of addition of heparin, indicating that heparin is needed to facilitate the formation of fibrillation nuclei. Aggregation is completely inhibited by submicellar concentrations of zwitterionic and anionic surfactants. Unlike previous reports, our NMR data do not indicate persistent structure in the presence of zwitterionic surfactant micelles. Thus kisspeptin can aggregate under physiologically relevant conditions provided heparin is present, but the process is highly sensitive to the presence of amphiphiles, highlighting the very dynamic nature of the peptide conformation and suggesting that kisspeptin aggregation is a biologically regulatable process. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 678–689, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Impact of α‐hydroxy‐propanodeoxyguanine adducts on DNA duplex energetics: Opposite base modulation and implications for mutagenicity and genotoxicity

Acrolein is an α,β‐unsaturated aldehyde that is a major environmental pollutant, as well as a product of cellular metabolism. DNA bases react with acrolein to form two regioisomeric exocyclic guanine adducts, namely γ‐hydroxy‐propanodeoxyguanosine (γ‐OH‐PdG) and its positional isomer α‐hydroxy‐propanodeoxyguanosine (α‐OH‐PdG). The γ‐OH‐PdG isomer adopts a ring‐opened conformation with minimal structural perturbation of the DNA host duplex. Conversely, the α‐OH‐PdG isomer assumes a ring‐closed conformation that significantly disrupts Watson‐Crick base‐pair alignments within the immediate vicinity of the damaged site. We have employed a combination of calorimetric and spectroscopic techniques to characterize the thermodynamic origins of these lesion‐induced structural alterations. Specifically, we have assessed the energetic impact of α‐OH‐PdG centered within an 11‐mer duplex by hybridizing the adduct‐containing oligonucleotide with its complementary strand harboring a central base N [where N = C or A], yielding a pair of duplexes containing the nascent lesion (α‐OH‐PdG·C) or mismatched adduct (α‐OH‐PdG·A), respectively. Our data reveal that the nascent lesion is highly destabilizing, whereas its mismatched counterpart partially ameliorates α‐OH‐PdG‐induced destabilization. Collectively, our data provide energetic characterizations of the driving forces that modulate error‐free versus error‐prone DNA translesion synthesis. The biological implications of our findings are discussed in terms of energetically probing acrolein‐mediated mutagenicity versus adduct‐induced genotoxicity. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 370–382, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

High‐resolution structures of collagen‐like peptides [(Pro‐Pro‐Gly)4‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)4]: Implications for triple‐helix hydration and Hyp(X) puckering

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

VCD spectroscopic properties of the β‐hairpin forming miniprotein CLN025 in various solvents

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the β‐hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable β‐hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a β‐hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave‐assisted solid phase peptide synthesis with N^α‐Fmoc protected amino acids. The VCD spectra displayed a (?,+,?) pattern with bands at 1640 to 1656 cm^?1, 1667 to 1687 cm^?1, and 1679 to 1686 cm^?1 formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm^?1 with component bands at 1630 cm^?1, 1646 cm^?1, 1658 cm^?1, and 1675 to 1680 cm^?1 that are indicative of the β‐sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of β‐hairpins formed by ^DPro‐Xxx turns and indicated that observed pattern is typical of β‐hairpins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 442–450, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Differential effects of phe19 and phe20 on fibril formation by amyloidogenic peptide Aβ16–22 (Ac‐KLVFFAE‐NH2)

The sequence KLVFFAE (Aβ16–22) in Alzheimer's β‐amyloid is thought to be a core β‐structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in β‐sheet. To elucidate the mechanism of the initial folding process, we undertook combined X‐ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized Aβ16–22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild‐type (WT) Aβ16–22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab‐like β‐crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30‐Å period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side‐chain dispositions for WT and 20CN, but not for 19CN. These X‐ray results and modeling imply that in the assembly of WT Aβ16–22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. Proteins 2010. © 2010 Wiley‐Liss, Inc.