Investigation of bifunctional chemical crosslinking reagents for studying 3D structures of biomolecules by mass spectrometry

Evaluating the 3D structure of biomolecules, such as proteins, nucleic acids, and their complexes is crucial to understand the relationship between their structure and function. Currently, x-ray crystallography and NMR spectroscopy are the primary techniques used for studying topological organization of biomolecules. However, obtaining good quality crystals is the bottleneck of the x-ray investigation and NMR has significant size limitations. A possible alternative requires employing crosslinking reagents in combination with mass spectrometry (MS) to identify the contacts between nucleic acid or protein components. This method has been called mass spectrometry 3-dimensional (MS3D). This research dissertation started with the evaluation of existing crosslinking reagents as possible MS3D probes and established the baseline conditions for the application of structural probes. Different classes of reagents were taken into consideration including nitrogen mustards, platinum compounds, 1,2-dicarbonyl compounds, triazines and some other synthetic antitumor agents. After baseline reaction conditions were established for the various crosslinking reagents, we demonstrated that MS3D could provide valid structure information using a fully characterized RNA substrate from HIV-1, SL1A. Then we tackled two RNA peudoknots, VPK and FIV-PK, which play important roles in ribosomal frameshifting. Nitrogen mustard (NM) was used as the main crosslinking reagent to obtain valid spatial constraints. All-atom models were generated for both VPK and FIV-PK: the MS3D model of the former compared very favorably with the corresponding NMR structure, providing only an average of 3 ? root mean square deviation. In the absence of a high-resolution structure, the model of FIV-PK constitutes the first example of an unknown 3D structure solved exclusively by MS3D. To expand the MS3D database with reagents manifesting a wide range of target specificity, arginine specific reagents were also developed. The arginine specificity and crosslinking ability of the developed reagents were confirmed by their application on three model proteins. Finally, we described our efforts in developing novel hetero-bifunctional reagents for specifically targeting protein-nucleic acids complexes. We selected metal chelates as the functional group with specificity toward the phosphate groups of nucleic acids, epichlorohydrin as the second reactive group of the hetero-functional crosslinker, which was intended to target cognate proteins. Our data showed that IDA-Ga3+ and IDA-Al3+ bound most tightly to DNA substrates than other metal chelates. The coupling of the second function group to the metal chelate was readily performed and the coupled bifunctional reagent retained DNA binding ability