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Electrospray ionization (ESI) mass spectrometry (MS) isThe globular or braided topology with attendant surface crevices and interior cavities creates a unique three-dimensional structure for proteins and, together with their dynamic properties mediated through local fluctuations or large-scale conformational changes, the singular functions of these lifesustaining biomolecules are executed. Conformations and conformational dynamics are key to the protein's functional integrity and these characteristic properties are highly dependent on environmental conditions. Thus, varying physiological conditions may play an important role in protein conformational changes. For example, pH adjustments in organisms can drive ligand-receptor dissociation and receptor recycling (1). Protein conformational changes are also manifest in the protein life cycles from expression to final post-activity phase, i.e. birth, function, and death (2), and improper protein conformations may be responsible for a number of diseases (3). Detailed information is required on protein higher-order structures and dynamics to fully understand how proteins perform their biological functions at the molecular level.Protein conformational changes can be monitored by conventional biophysical and analytical methods, including circular dichroism (CD) 1 (4), tryptophan fluorescence (5), and infrared spectroscopy (6). Far-ultraviolet (UV) CD spectroscopy, for instance, provides information mainly on secondary structural elements of the polypeptide chains; near-UV-CD detects changes in the tertiary structure around aromatic amino acid side chains, and fluorescence techniques may be another more sensitive technique for this purpose. The low-resolving power inherent in these methods, however, limits the investigator's ability to focus on regions where conformational changes might occur. Detailed structural information at individual residues of a protein can, of course, be obtained from From the

A four-stranded DNA junction (Fig. 1a) was first proposed by Robin Holliday in 1964 as a structural intermediate in a mechanistic model to account for the means by which genetic information is exchanged in yeast (1). This mechanism for genetic exchange is now generally known as homologous recombination and the four-stranded intermediate as the Holliday junction. The general mechanism for recombination has undergone a number of revisions in detail, but the Holliday junction remains a key component in the process and in a growing number of analogous cellular mechanisms (reviewed in Refs. 2 and 3), including site-specific recombination (4), resolution of stalled replication forks (5, 6), DNA repair (7,8), and phage integration (9). Consequently, the structural and dynamic properties of the Holliday junction have been the focus of intense study since the mid-1960s. As is often the case in science, a multitude of single crystal structures of Holliday junctions in various forms have been solved over a relatively short period of time but only after decades of disappointment. The first structures of junctions in complexes with recombination and DNA repair proteins were reported in 1997 and 1998 (10, 11), whereas junctions in RNA-DNA complexes (12, 13) and in DNA-only constructs (14, 15) emerged just as the twentieth century came to a close. In this review, we will focus on the structures of DNA-only junctions and their geometries, as defined by sequence and ion-dependent interactions. BC (Before Crystal) StructuresBefore any of the crystal structures of junctions were available, the general architecture and dynamic properties of the Holliday junction were already being defined through biochemical and biophysical studies on immobilized junctions assembled from asymmetric sequences (reviewed in Ref. 2). From comparative gel and solution studies, the junction was known to undergo ion-dependent conformational transitions. In low salt solutions, the junction adopts an extended "open-X" form to minimize the strong repulsion between negatively charged phosphates at the crossover strands that exchange between DNA double helices (Fig. 1b). The junction collapses to a stacked-X form when the phosphates are screened by cations, particularly by magnesium (Mg 2ϩ ) and other divalent ions. This more compact junction pairs the arms into coaxially stacked, semicontinuous duplexes, with each stacked duplex having an outside non-exchanging strand and an inside crossover strand. The crossed-over strands can be oriented either in parallel or antiparallel directions.Although the parallel configuration of the stacked-X junction (Fig. 1a) has been suggested to be formed by extrusion of cruciform structures in negatively supercoiled closed circular DNAs (16,17) and in synthetic DNA arrays (18), it is the antiparallel form that is seen by gel (19 -21) and, subsequently, fluorescence resonance energy transfer studies (22-24) and by atomic force microscopy in interlinked sheets of DNA (25, 26). The basic model that emerged for the antiparallel st...

Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) participates in rheumatoid arthritis (RA) pathogenesis by facilitating leukocyte infiltration, however, its other pathological functions are not fully defined in RA. In the present study, we evaluated the effect of RANTES/CCL5 on tissue degrading enzymes matrix metalloproteinase-1 (MMP-1) and MMP-13 expression and its contribution to the progressive joint damage by RA synovial fibroblasts (RASFs). Our results showed that RANTES/CCL5 dose dependently induced MMP-1 and MMP-13 expression in monolayers and three-dimensional (3D) micromass of human RASFs, which correlated with an increase in collagenase activity. This activation by RANTES/CCL5 was observed in RASF, but not in osteoarthritis SFs (OASFs). Evaluation of the signaling events showed that RANTES/CCL5 selectively activated PKCδ, JNK, and ERK proteins to induce MMP expression in human RASFs. Pretreatment with a functional antagonist (Met-RANTES) or heparinase III [an enzyme that selectively digests heparan sulfate proteoglycans (HSPGs)] completely abrogated RANTES/CCL5-induced MMP-1 and MMP-13 expression. Interestingly, the inhibition of RANTES/CCL5 using small-interfering RNA approach reduced the ability of interleukin-1β (IL-1β) to induce MMP-1 and MMP-13 expression, asserting its mediatory role in tissue remodeling. In the inhibitor study, only the selective inhibition of HSPGs or PKCδ, ERK, and JNK markedly inhibited RANTES/CCL5-induced MMP-1 and MMP-13 production. Circular dichroism spectroscopy results demonstrated the degradation of collagen triple-helical structure upon exposure to the conditioned media from RANTES/CCL5 stimulated RASFs, which was reverted by a broad-spectrum MMP inhibitor (GM6001). These findings suggest that RANTES/CCL5 not only upregulates MMP-1 and MMP-13 expression by partly utilizing HSPGs and/or PKCδ-JNK/ERK pathways but also mediates IL-1β-induced MMP-1 and MMP-13 expression.

A number of single-crystal structures have now been solved of the four-stranded antiparallel stacked-X form of the Holliday junction. These structures demonstrate how base sequence, substituents, and drug and ion interactions affect the general conformation of this recombination intermediate. The geometry of junctions had previously been described in terms of a specific set of parameters that include: (i) the angle relating the ends of DNA duplexes arms of the junction (interduplex angle); (ii) the relative rotation of the duplexes about the helix axes of the stacked duplex arms (J(roll)); and (iii) the translation of the duplexes along these helix axes (J(slide)). Here, we present a consistent set of definitions and methods to accurately calculate each of these parameters based on the helical features of the stacked duplex arms in the single-crystal structures of the stacked-X junction, and demonstrate how each of these parameters contributes to an overall conformational feature of the structure. We show that the values for these parameters derived from global rather than local helical axes through the stacked bases of the duplex arms are the most representative of the stacked-X junction conformation. In addition, a very specific parameter (J(twist)) is introduced which relates the relative orientation of the stacked duplex arms across the junction which, unlike the interduplex angle, is length independent. The results from this study provide a general means to relate the geometric features seen in the crystal structures to those determined in solution.