Crystallographic studies of an anti-ssDNA Fab and PutA proteins

Protein function and structure are intimately related, and X-ray crystallography is the most robust method for elucidating three-dimensional structure. This dissertation discusses technical aspects of using sulfur anomalous scattering and twinned crystals in protein crystallography. These methods are then demonstrated in the structure determinations of an anti-single-stranded DNA antigen-binding fragment and a proline catabolic enzyme.; The use of single-wavelength anomalous dispersion (SAD) from S atoms collected in-house to overcome model bias in molecular-replacement (MR) structure determination is demonstrated. The test case considered is a P6(5)22 anti-ssDNA Fab crystal with a theoretical anomalous signal of 0.8% and a diffraction limit of 2.3 A, from which a 360 degrees, 39-fold redundant data set was collected. A nearly complete anomalous scatterer substructure could be quickly built from anomalous difference Fourier analysis based on phases from a full or partial MR solution. The resulting SAD phases were improved with density modification and used to calculate an unbiased electron-density map that could be used for model building. This map displayed clear and continuous density for almost the entire main chain, as well as good density for most side chains. The favorable results obtained from this realistic test case suggest that anomalous differences from S atoms should be routinely collected and used in MR structure determination.; Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. This dissertation reports the structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid), as well as two structures of the unliganded Fab. One of the unliganded Fab structures was determined using data from a merohedrally-twinned crystal, which presented unique challenges. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, HIM, and 1-H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison of the DNA-1/HEPES structure to the previously solved DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). The two structures of unliganded DNA-1 suggest that HCDR3 becomes ordered upon complex formation. This work implicates structural plasticity of HCDR3 as critical for ligand recognition by DNA-1.; The last project focuses on the proline utilization protein A (PutA) protein, which is a bacterial flavoprotein that catalyzes the two-step oxidation of proline to glutamate. In enteric bacteria, PutA also functions as a transcriptional repressor of the putA and putP genes. This dissertation presents the first crystal structure of a full-length PutA protein.