Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p)

Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using 14N/15N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers

Quantitative Mass Spectrometric Analysis of RNA-Protein Cross-Links

In recent times much emphasis has been laid on revealing the composition and regulation of various RNP complexes. The structural studies of the RNP complexes provide a valuable insight into the binding modes and functional implications of interactions between the RNAs and RNA binding proteins within the complexes. To investigate the interactions of the RNA-binding proteins within RNP complexes, UV-induced cross-linking followed by mass spectrometry (MS) has proved to be a promising and straightforward technique. But the limitations of most of the purification methods as well as the intricate mass spectrometric data analysis have hampered the study of these RNP complexes. During the course of this study, the protocol was modified and optimized for the interaction analysis of large RNP complex assemblies like RNP complexes isolated from the HeLa nuclear extract which led to the identification of predicted as well as unknown RBMs. Moreover, the qualitative analysis of the protein-RNA cross-links derived from in vitro assembled Brat-NHL-hb RNA complex and CWC2-U6/U4 snRNAs complexes was also carried out. Based on the qualitative analysis of CWC2-U6/U4 cross-links, the quantitative analysis of protein-RNA cross-links has been established. The studies conducted during the research work have contributed in the identification and characterization of protein-RNA interactions within the aforementioned complexes and also provided the quantitative insight into the protein-RNA interactions.2020-03-0

Protein complexes in chlorophyll biosynthetic enzymes

Proteins are found on the inside, in the membrane, on the surface and on the outside of cells. They form complicated structures and they interact with other molecules and proteins. Protein complexes and protein-protein interactions are challenging to investigate and in the beginning of protein research most studies were done with single proteins, often in water. Although, in vivo proteins rarely function alone.To study protein complexes, two enzymes in the chlorophyll biosynthetic pathway were selected, Mg-chelatase in Rhodobacter capsulatus (bacteria) and the MPE cyclase complex in Hordeum vulgare (barley) and Arabidopsis thaliana (mouse-ear cress). Chlorophyll is a pigment formed through a complicated reaction path. Chlorophyll biosynthesis takes place in chlorophyll-producing organisms. The firstcommitted step towards chlorophyll biosynthesis is performed by the enzyme complex Mg-chelatase. Mg-chelatase inserts a Mg2+ ion into the porphyrin substrate. The pathway is continued by a methyltransferase and thereafter the MPE cyclase complex which performs a complicated ring-closure in the porphyrin.Mg-chelatase is composed of three proteins, BchI (40 kDa), BchD (60 kDa) and BchH (130 kDa). A cryo-electron microscopy model of the BchID complex (7.5 Å) revealed a two-tired hexameric ring structure with an arrangement of the subunits as a trimer of dimers. The transient full complex of Mg-chelatase, BchIDH, was chemically cross-linked and BchH was found to interact with the Dside of the BchID complex.The MPE cyclase complex was more difficult to study and two of the three core components of the complex are still unknown. An interesting enzyme, NADPH-dependent thioredoxin reductase C (NTRC), was found to stimulate the MPE cyclase reaction together with a 2-Cys peroxiredoxin. NTRC was characterised further with regards to function and structure. The enzyme consists of a fusion between a NADPH-dependent thioredoxin reductase polypeptide and a thioredoxin polypeptide in the C-terminal. The three-dimensional structure ofNTRC was determined with cryo-electron microscopy (10.0 Å) and revealed a tetramer

In-Depth Analysis of Zero-Length Crosslinking for Structural Mass Spectrometry

The completion of the Human Genome Project revealed the sequence identity of essentially every human protein. However, in most cases, amino acid sequences alone convey little implication on the protein static structures, its dynamic conformational changes, and most importantly, its functions. To fully understand the behaviors and properties of macromolecular complexes, solving their 3D structures is necessary and highly critical. Under this rationale, structural genomics collaborations were initiated aiming to determine high-resolution structures of as many proteins and protein folds as possible, relying mostly on X-ray crystallography and NMR spectroscopy. Yet, very large, highly flexible or disordered, and dynamic protein complexes can exceed the capabilities of these high-resolution techniques. Although computational molecular modeling can be utilized, such structures are highly speculative and often inaccurate unless supported by actual experimental data. Structural mass spectrometry recently emerged as an alternative method which can provide medium-resolution spatial information capable of complementing computational approaches, and are applicable to heterogeneous samples with potentially no limit on complex sizes. In particular, chemical crosslinking coupled with mass spectrometry, has recently received considerable interest. Most recent progress focused on developing crosslinkers with special properties such as enrichment tags, isotopic labeling sites, or MS-cleavable bonds along with accompanying data analysis strategies and software packages. These crosslinkers insert their spacer arm between proximal amino acid residues, greatly reducing the stringency of the derived distance constraints. In contrast, zero-length crosslinkers are crosslinks which do not add any extra atoms to the product crosslinked peptides, therefore providing the tightest possible spatial constraints but rendering enrichment and isotopic labeling strategies inapplicable. As a result, zero-length crosslinking received limited attention and no software tools have previously been specifically developed for it. In this thesis project, we developed a multi-tiered mass spectrometry data acquisition and computational data analysis strategy along with a dedicated software tool to enhance identification of zero-length crosslinks in complex samples. Label-free comparison and targeted high-resolution mass spectrometry were utilized to filter out the vast majority of non-crosslinked peptides and increase confidence of crosslink identification, compensating for the lack of enrichment techniques and characteristic MS patterns employed by non-zero-length crosslinking methods. Each step from mass spectrometer acquisition parameters to MS/MS spectra evaluation functions was optimized based on zero-length crosslinking datasets of proteins with known crystal structures. Our pipeline was then applied to probe structures and conformational changes of mini-spectrin, a 90 kDa recombinant protein that closely mimics erythrocyte spectrin\u27s dynamic dimer-tetramer equilibrium. Compared to previous analyses performed in our laboratory, the current strategy more than doubled the number of identified crosslinks and significantly reduced analysis time per experiment from months to just several days. Distance constraints derived from mini-spectrin crosslinks were used as inputs in subsequent homology modeling, allowing development of experimentally-verified medium-resolution structures for wild-type mini-spectrin tetramer and both wild-type and hereditary elliptocytosis (HE) mutant mini-spectrin dimers. The structure models, in combination with independent biophysical experiments, illustrated how such distal HE-related mutations destabilized spectrin dimer-tetramer equilibrium by simultaneously lowering thermal stability of tetramer and giving rise to a more-compact, more-stable closed dimer conformation

Implementation of nano-liquid chromatography hyphenated to tandem mass spectrometry for protein identification in gel and non-gel based proteomics

This thesis is divided into three parts. Part I deals with the current status of large scale strategies for the analysis of proteins in biological systems. In Chapter I, genomics and the need to shift towards proteomic approaches are outlined. An overview of key technologies used in functional and structural proteomics is provided in Chapter II, whereas mass spectrometry and the strategies for profiling of proteins are discussed in detail in the final chapter of this part (Chapter III). Part II describes the analysis of cytosolic proteins by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. First, the techniques are highlighted that were used to identify gel separated proteins, i.e. the implementation of nano-liquid chromatography and mass spectrometry (Chapter IV). The next two chapters are applications of the developed methodology in two case studies: the protein composition of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 grown on ferric oxide (Chapter V) and the effect of a short-term heat shock on the plant barley (Chapter VI). Part III deals with the development of alternative strategies for the analysis of membrane proteins. A special electrophoretic technique, i.e. blue-native polyacrylamide gel electrophoresis, in combination with mass spectrometry, was used for profiling the different subunits of the oxidative phosphorylation system (Chapter VII), while multi-dimensional liquid chromatography coupled to MALDI tandem mass spectrometry was used for the profiling of the proteins present in the murine myelin sheath (Chapter VIII)

Mass spectrometry approaches for profiling protein-protein interactions

This dissertation is focused on developing cross-linking and mass spectrometry methodologies to study protein-protein interactions. Top-down cross-linking, in combination with mass spectrometry, provides advantages over bottom-up approaches, such as retaining posttranslational modification. Intermolecular cross-linking studies focus on defining protein complex topology and protein-protein interactions. We first developed the top-down MS approach to analyze intermolecular cross-linking in human hemoglobin. Both α-α and β-β intermolecular cross-linking were found and the cross-linking sites on the protein were identified, obtaining distance constraints between subunits of the human hemoglobin protein complex. This methodology would be a promising approach to characterize protein complexes and protein-protein interactions with high throughput and automation. This dissertation also focuses on development of cross-linking mass spectrometry to study synphilin-1 interactors and aggresome formation. Synphilin-1 is a protein that promotes the formation of protein aggregates and aggresome formation upon proteasome inhibition, and is implicated in Parkinson disease. Synphilin-1 contains several protein binding motifs. The biological functions of synphilin-1 and its role in aggresome formation and the pathogenesis of Parkinson disease remain to be elucidated. We utilized tandem affinity purification and label-free mass spectrometry to explore the patterns of cellular proteins associated with synphilin-1. We identified 57 synphilin-1 interacting proteins, and functional enrichment and pathway analysis showed that many of the associated proteins are involved in chromatin modulation, RNA and protein metabolism. Furthermore, we developed a proteomic strategy to identify synphilin-1 binary interacting partners as well as interacting domains using affinity purification followed by isotopically tagged cross-linking in combination with mass spectrometry. We found 24 newly discovered proteins that directly bind to synphilin-1. The proteins were mainly involved in RNA metabolism. The coiled-coil domain (CC), ankyrin-like repeat domain 2 (ANK2), and the protein aggregate promoting domain, appeared to the main regions that bound proteins. The functions of synphilin-1 interacting proteins, such as CK2, in aggresome formation were studied. The results show that CK2 is an important regulator of aggresome formation, but not through its kinase activities. Involvement of synphilin-1 in autophagy was also investigated. Knockdown of synphilin-1 shows that synphilin-1 impacts autophagy