Protein folds have been invented and adopted under various evolutionary constraints. The question arises how general mechanical attributes that control protein function have been sampled by the evolving protein domain repertoire in the varying biological environment. We assessed three measures, namely flexibility, foldability, and mechanical strength, for ~100,000 protein domains, using geometrical sampling (Con- coord), contact order, and coarse-grained protein unfolding, respectively. In general, we find high mechanical stability to involve low protein flexibility and low foldability. In order to reconstruct protein architecture evolution, we used infor-mation em- bedded in a structural genomic census of fold architectures defined by a phylo- genomic analysis of 185 completely sequenced genomes using advanced hidden Markov models. Interestingly, flexibility is found to increase in evolu- tion, hinting towards a fine-tuning of protein function during evolution towards dynamic regulation. Finally, our work can explain how proteins as an intricate network of inter-molecular bonds were designed by evolution as a response to mechanical stress. 1274-Pos Board B44 Ligand Recognition of Lipocalin-Type Prostaglandin D Synthase Shigeru Shimamoto 1,2 , Takuya Yoshida 2 , Yuya Miyamoto 3 , Takashi Inui 3 , Kosuke Aritake 4 , Yoshihiro Urade 4 , Tadayasu Ohkubo 2 . 1 Kinki University, Higashi-osaka, Japan, 2 Osaka University, Suita, Japan, 3 Osaka Prefecture University, Sakai, Japan, 4 Osaka Bioscience Institute, Suita, Japan. Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi functional protein that functions as a PGD 2 synthesizing enzyme, a scavenger of various lipophilic ligands, and an amyloid b chaperone in the brain. L-PGDS is a mem- ber of the lipocalin superfamily and has the ability to bind various lipophilic molecules, such as prostanoid, retinoid, bile pigment, and amyloid b peptide. However, the molecular mechanism for this wide variety of ligand binding is not fully understood. In this study, we determined the NMR structures of re- combinant mouse L-PGDS and a L-PGDS/PGH 2 analog. L-PGDS has a typical lipocalin fold, consisting of an eight-stranded b-barrel and a single a-helix. The interior of the barrel forms a hydrophobic cavity and the upper end of the barrel is open. The size of the barrel was found to be larger than those of other lipo- calins and the cavity contained two pockets. The results of NMR titration, ki- netic and molecular docking experiments revealed that PGH 2 and retinoic acid occupied the hydrophilic pockets 1 and 2, respectively. A structural comparison of the L-PGDS/PGH 2 analog complex with apo-L-PGDS showed that the H2- helix, CD-loop, and EF-loop located at the upper end of the b-barrel undergo a conformational change and cover the entrance of the cavity upon U-46619 binding. These results indicate that the two binding sites in the large cavity are responsible for the broad ligand specificity of L-PGDS in its induced-fit mechanism. 1275-Pos Board B45 Studying the Structure and Function of the Cold Shock Related Protein CspE, in Escherichia Coli through Molecular Modeling Andre’ Samuel, Nancy Trun, Ph.D. Duquesne University, Pittsburgh, PA, USA. In the bacterium Escherichia coli, a single 4,639 base pair circular chromo- some is compacted into about 100 independently supercoiled domains that will form a rosette structure, referred to as the nucleoid. One of the proteins shown to be involved in chromosome condensation is a 7-kDa protein called CspE. Escherichia coli K12, expresses nine Csp (Cold shock-related protein) gene products: (CspA-I) and they share between 29 and 83 percent identity in their amino acid sequences. The bacterium Bacillus subtilis has three Csp homologues with greater than 70% identity to CspE and studies have shown that Csp proteins bind to single stranded poly d(T) with a minimum of 6 thymines in an 8 base sequence. CspB in Bacillus subtilis has solved crystal structures both with and without it’s DNA ligand. While the crystal structure of CspA from Escherichia coli has been elucidated, the remaining eight Csps have not. This work describes the use of structural information from the homologue CspB in B. subtilis to model the structure of the remaining eight unsolved structures of the nine Csps in E. coli. A single point mutation in the computationally predicted binding site of CspE results in the loss of its DNA binding function while a single point mutation outside of the predicted binding site shows no significant difference in DNA binding compared to wildtype. 1276-Pos Board B46 Probing the Nickel Coordination Environment in Helicobacter Pylori NikR Sarah Bowman, Catherine L. Drennan, Collin M. Stultz. MIT, Cambridge, MA, USA. Nickel homeostasis in gram-negative bacteria is often regulated at the transcrip- tional level by the nickel-dependent protein NikR, which belongs to the ribbon- helix-helix DNA-binding protein family. Generally, NikR is found as a homote- tramer, with four nickel ions bound of the tetrameric interface at a metal binding domain (MBD). In Helicobacter pylori, NikR (HpNikR), unlike homologous NikR proteins, sits at a regulatory hub and has been found to have a variety of regulatory roles. HpNikR has been implicated in the regulation of proteins that function in acid acclimation, nickel uptake, virulence, and stress response. There are a number of questions about the nickel coordination environment in HpNikR due to a series of puzzling and sometimes contradictory structural and experimental results. Here we present results from complimentary compu- tational and experimental techniques to help further resolve these questions. Spe- cifically, we use molecular dynamics simulations and spectroscopic experiments to investigate the nickel coordination environment. We use circular dichroism to observe differences in the thermal stability of HpNikR depending on the nickel:- protein stoichiometry, with stabilization increasing as nickel concentration in- creases. Stability of HpNikR is found to be pH dependent. Elucidation of details about nickel coordination in HpNikR will improve our understanding of the functional role of genetic regulatory pathways in H. pylori pathogenicity. 1277-Pos Board B47 Toward Unraveling the Structure and Molecular Basis of Ferrous Iron Transport of Feo from Klebsiella Pneumoniae Tai-huang Huang, Kuo-Wei Hung, Yi-Wei Chang, Chwan-Deng Hsiao. Academia Sinica, Taipei, Taiwan. Feo from K. pneumoniae is a unique type of ferrous iron transporter for bacte- ria. It consists of three proteins, FeoA, a small SH3-domain like protein; FeoB, a large protein consisting of a cytosolic N-terminal G-protein domain and a C-terminal transmembrane domain that may serve as a Fe þþ permease; and FeoC, a small putative transcriptional factor. Using X-ray crystallography and NMR we have solved the structure of FeoA, the intracellular domain of FeoB (NFeoB), FeoC and NFeoB/FeoC complex. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-do- main), which despite its lack of sequence similarity between species is structur- ally conserved. In the nucleotide-free state, the G domain’s two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide-binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays impor- tant roles in FeoB function. The structures and their functional implications of FeoA, FeoC and NFeoB/FeoC complex will be discussed also. 1278-Pos Board B48 Structural Basis of the Specific Functions of Proteins Secreted from Mam- mary Gland during Different Progressive Stages such as Proliferative, Early-Lactation, Late-Lactation and Involution Phases Ravi V. Santani, Sanket Kaushik, Amit Kumar Singh, Pradeep Sharma, Mau Sinha, Sujata Sharma, Tej Pal Singh. AIIMS, New Delhi, India. Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India-110029 The mammary gland undergoes repeated cycles of pregnancy, lactation and in- volution. Throughout these cycles, the interactions of mammary gland epithe- lial cells with their surrounding extracellular matrix (ECM), in particular the specialized ECM known as basement membrane (BM), contributes to the sig- nals required for their proliferation, differentiation and survival. The structural and functional studies of prominent proteins secreted during these various phases have been carried out. The proteins included in these studies are insulin growth factor (IGF), lactoferrin (LF), Lactoperoxidase (LP), peptidoglycan rec- ognition protein (PGRP) and signaling glycoproteins (SPX-40). Three- dimensional structural determinations using X-ray crystallographic methods 252a Monday, February 27, 2012