Self-assembly of left- and right-handed molecular screws.

Stereoselectivity is a hallmark of biomolecular processes from catalysis to self-assembly, which predominantly occur between homochiral species. However, both homochiral and heterochiral complexes of synthetic polypeptides have been observed where stereoselectivity hinges on details of intermolecular interactions. This raises the question whether general rules governing stereoselectivity exist. A geometric ridges-in-grooves model of interacting helices indicates that heterochiral associations should generally be favored in this class of structures. We tested this principle using a simplified molecular screw, a collagen peptide triple-helix composed of either l- or d-proline with a cyclic aliphatic side chain. Calculated stabilities of like- and opposite-handed triple-helical pairings indicated a preference for heterospecific associations. Mixing left- and right-handed helices drastically lowered solubility, resulting in micrometer-scale sheet-like assemblies that were one peptide-length thick as characterized with atomic force microscopy. X-ray scattering measurements of interhelical spacing in these sheets support a tight ridges-in-grooves packing of left- and right-handed triple helices.

Circular permutation directs orthogonal assembly in complex collagen peptide mixtures.

Multiple types of natural collagens specifically assemble and co-exist in the extracellular matrix. Although noncollagenous trimerization domains facilitate the folding of triple-helical regions, it is intriguing to ask whether collagen sequences are also capable of controlling heterospecific association. In this study, we designed a model system mimicking simultaneous specific assembly of two collagen heterotrimers using a genetically inspired operation, circular permutation. Previously, surface charge-pair interactions were optimized on three collagen peptides to promote the formation of an abc-type heterotrimer. Circular permutation of these sequences retained networks of stabilizing interactions, preserving both triple-helical structure and heterospecificity of assembly. Combining original peptides A, B, and C and permuted peptides D, E, and F resulted primarily in formation of A:B:C and D:E:F, a heterospecificity of 2 of 56 possible stoichiometries. This degree of specificity in collagen molecular recognition is unprecedented in natural or synthetic collagens. Analysis of natural collagen sequences indicates low similarity between the neighboring exons. Combining the synthetic collagen model and bioinformatic analysis provides insight on how fibrillar collagens might have arisen from the duplication of smaller domains.

Osteogenesis imperfecta missense mutations in collagen: structural consequences of a glycine to alanine replacement at a highly charged site.

Glycine is required as every third residue in the collagen triple helix, and a missense mutation leading to the replacement of even one Gly in the repeating (Gly-Xaa-Yaa)(n) sequence with a larger residue leads to a pathological condition. Gly to Ala missense mutations are highly underrepresented in osteogenesis imperfecta (OI) and other collagen diseases, suggesting that the smallest replacement residue, Ala, might cause the least structural perturbation and mildest clinical consequences. The relatively small number of Gly to Ala mutation sites that do lead to OI must have some unusual features, such as greater structural disruption because of local sequence environment or location at a biologically important site. Here, peptides are used to model a severe OI case in which a Gly to Ala mutation is found within a highly stabilizing Lys-Gly-Asp sequence environment. Nuclear magnetic resonance, circular dichroism, and differential scanning calorimetry studies indicate this Gly to Ala replacement leads to a substantial loss of triple-helix stability and nonequivalence of the Ala residues in the three chains such that only one of the three Ala residues is capable of forming a good backbone hydrogen bond. Examination of reported OI Gly to Ala mutations suggests their preferential location at known collagen binding sites, and we propose that structural defects caused by Ala replacements may lead to pathology when they interfere with interactions.

Computational design of a collagen A:B:C-type heterotrimer.

We have successfully designed an A:B:C collagen peptide heterotrimer using an automated computational approach. The algorithm maximizes the energy gap between the target and competing misfolded states while enforcing a minimum target stability. Circular dichroism (CD) measurements confirm that all three peptides are required to form a stable, structured triple helix. This study highlights the power of automated computational design, providing model systems to probe the biophysics of collagen assembly and developing general methods for the design of fibrous proteins.

Self-association of streptococcus pyogenes collagen-like constructs into higher order structures.

A number of bacterial collagen-like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple-helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold-shock expression system is used to obtain purified recombinant collagen-like protein (V-CL) from Streptococcus pyogenes containing an N-terminal globular domain V followed by the collagen triple-helix domain CL and the modified construct with two tandem collagen domains V-CL-CL. Both constructs and their isolated collagenous domains form stable triple-helices characterized by very sharp thermal transitions at 35-37 degrees C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V-CL-CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL-CL, form units of diameter 4-5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen-like protein is a cell surface protein with no indication of participation in higher order structure, the triple-helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.

Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline.

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)(79), which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)(n) sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 degrees C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 degrees C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.

Neurolisteriosis en adultos: a propósito de seis casos clínicos / Neurolisteriosis in adults: report of six clinical cases

INTRODUCCION: Listeria monocytogenes tiene una especial predilección por infectar el sistema nervioso central y sus cubiertas meningeas. Afecta a pacientes que se encuentran en edades extremas de la vida, pacientes con deficiencia en su inmunidad celular y adultos sanos. La forma mas común de manifestarse es la meningitis aguda, aunque puede expresarse como cerebritis, encefalitis de tronco (romboencefalitis), y excepcionalmente mielitis. CASUISTICA: Se presentan y comentan seis casos clinicos de neurolisteriosis, cinco en adultos sanos, con sus hallazgos imagenológicos y licuorales. RESULTADOS: Tres de los pacientes se presentaron como meningitis aguda, uno como meningoencefalitis, otro como cerebritis y el restante como romboencefalitis. Se destaca el carácter turbio o ligeramente turbio del líquido cefalo-raquideo (LCR), la glucorraquia normal detectada en tres de los casos y el diagnostico realizado en cinco de los casos por cultivo del LCR. Se comenta la resonancia magnética singular del caso de la romboencefalitis con microabscesos en tronco. Todos los pacientes tuvieron evolución satisfactoria con tratamiento antibiotico. CONCLUSION: La neurolisteriosis debe ser un diagnostico a tener en cuenta no solo en pacientes inmunocomprometidos o en edades extremas de la vida. Debe también tenerse en cuenta en pacientes adultos jóvenes sanos procedentes de regiones donde las condiciones sanitarias son precarias y no existe un adecuado control en la elaboración de alimentos.

Effect of deamidation on stability for the collagen to gelatin transition.

Deamidation of amide residues, Asn and Gln, in collagen occurs during the manufacture of B-type gelatin and could affect the performance of B-type gelatins as it may affect the refolding of triple-helical junctional domains that are formed during gelation. Host-guest peptides of the form acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide, where the X- and Y-positions of the guest peptide are varied, have been used to examine the effect of changing Asn to Asp and Gln to Glu on triple-helix stability. This paper reports the stability of host-guest peptides containing the guest triplets Gly-Ala-Asn, Gly-Asn-Ala, Gly-Asn-Lys, Gly-Gln-Ala, Gly-Glu-Glu, and Gly-Leu-Glu. In combination with previous data, these now provide 15 pairs of peptides in which the effect of deamidation can be compared. These comparisons show that the deamidation of Asn to Asp, regardless of whether it occurred in either the X- or Y-position, always gave a stabilizing effect; deamidation of Gln in the X-position also led to an increase in stability. In contrast, deamidation of Gln in the Y-position was quite distinct, leading to destabilization. The higher observed frequency of Gln in the Y-position compared with other amides may account for the slight destabilization of collagen following deamidation.

[Neurolisteriosis in adults: report of six clinical cases]. / Neurolisteriosis en adultos: a propósito de seis casos clínicos.

INTRODUCTION: Listeria monocytogenes shows a special attraction to infect the central nervous system and its meningeals coats. It affects newborn as well as elderly people, patients with deficiencies in their cellular immune systems, and healthy adults. It presents most commonly as an acute meningitis, although it can present itself as cerebritis, brain stem encephalitis (rhomboencephalitis), and exceptionally as myelitis. PATIENTS: We describe six clinical cases of neurolisterioris, five of which in healthy adults; we also describe the images and cerebrospinal fluid (CSF) findings, RESULTS: Three patients contracted acute meningitis, one of them meningoencephalitis, one cerebritis and the last one rhomboencephalitis. The CSF was turbid or slightly turbid with normal glycorrachia in three patients. The diagnosis was made in five through culture of CSF. The patient with the rhomboencephalitis had brain stem microabscesses in the brain magnetic resonance. All the patients had a good outcome under antibiotics treatment. CONCLUSION: Neurolisteriosis has to be kept in mind as a feasible diagnosis not only in immunocompromised but also in newborn as well as elderly patients. It should also be taken into account in young healthy adults living in regions under poor sanitary conditions where there is no adequate control of food manufacturing.

Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders.

Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.

Stepwise construction of triple-helical heparin binding sites using peptide models.

The specific localization of the asymmetric form of acetylcholinesterase (AChE) in neuromuscular junctions results from the interaction of its collagen-like tail with heparan sulfate proteoglycans in the synaptic basal lamina. This interaction involves two heparin binding consensus sequences of the form XBBXB, where B is a basic residue, located in the triple-helical collagen tail: GRKGR for the N-terminal site and GKRGK for the C-terminal site. To explore the basis of the higher heparin affinity seen for the C-terminal site vs. the N-terminal site, two homologous series of (Gly-Xaa-Yaa)(8) peptides were constructed to model these triple-helical binding sites. Individual tripeptide units from each heparin binding site were introduced in a stepwise fashion into a Gly-Pro-Hyp framework, until the consensus sequence and its surrounding triplets were recreated. As each additional triplet from the binding site is inserted to replace a host Gly-Pro-Hyp triplet, the triple-helix stability decreases, and the drop in thermal stability is close to that expected if each Gly-X-Y triplet contributed independently to global stability. CD spectroscopy and calorimetry show the stability of these AChE model peptides is increased by addition of heparin, confirming binding to heparin, and the lack of significant enthalpy change indicates the binding is largely electrostatic in nature. Displacement assays measure the strength of the peptide-heparin interaction, and indicate an inverse correlation between the peptide ability to bind heparin and its thermal stability. The model peptides for the C-terminal binding site show a greater heparin affinity than the peptide models for the N-terminal binding site only when residues surrounding the consensus sequence are included.