Matching cross-linked peptide spectra: only as good as the worse identification.

Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders. Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linker-specific ions is assessed. The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are analyzed. In addition, when FDR thresholds are calculated separately for intraprotein and interprotein results, a further improvement in the number of unique cross-links confidently identified is achieved. These improvements are demonstrated on two previously published cross-linking datasets.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II.

The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.

Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

Structure of the '30 nm' chromatin fibre: a key role for the linker histone.

The structure of the '30 nm' chromatin fibre has eluded us for 30 years and remains a major unsolved problem in biology. Progress during the past year has led to the proposal of two significantly different models: one derived from the crystal structure of a four-nucleosome core array lacking the linker histone and the other, much more compact structure, derived from electron microscopy analysis of long nucleosome arrays containing the linker histone. The first model is of the two-start helix type, the second a one-start helix with interdigitated nucleosomes. These models provide new evidence that the topology and compactness of the '30 nm' chromatin fibre structure are regulated by the linker histone. The structural information also provides insights into the mechanisms by which the degree of chromatin compaction might be regulated by histone composition and post-transcriptional modifications.

A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone.

An understanding of the role of higher-order chromatin structure in the regulation of cellular processes such as transcription will require knowledge of the structure of the "30 nm" chromatin fibre and its folding and unfolding pathways. We report an in vitro chromatin reconstitution system, which uses arrays of 12 and 19 copies of a 200 bp repeat of the Widom 601 DNA sequence. Since this DNA sequence binds the histone octamer with much higher affinity than mixed sequence DNA, we have used competitor DNA in the reconstitutions to control the loading of both the histone octamer and linker histone onto the 601 DNA arrays. Using this method we have obtained nucleosome arrays that have one histone octamer and one H5 bound per 200 bp repeat, and hence have the stoichiometric composition of native chromatin. To obtain highly compact 30 nm chromatin fibres, we have investigated a number of folding buffer conditions including varying NaCl or MgCl(2) concentrations. Sedimentation velocity analysis shows that the reconstituted nucleosome arrays have the same folding properties as native chromatin and form highly compact structures in high NaCl concentrations or 1mM MgCl(2). Negative stain and electron cryo-microscopy of the folded arrays show a homogeneous population of folded particles with a uniform diameter of 34 nm. The data presented provide good evidence that the reconstitution method we have developed produces, for the first time, a defined population of folded 30 nm fibres suitable for detailed structural investigation.

Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction.

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.

EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure.

Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.