Polysaccharide lyases (PLs) are enzymes that our commonly used by microorganisms to degrade polyuronides. These enzymes are produced by bacteria to aid in reducing the high molecular weight of the host's extracellular matrix to enhance its virulence. PLs can also be used to degrade polysaccharides as a source of carbon for the pathogen. Our focus is on the alginate lyase of Stenotrophomonas maltophilia (Smlt1473), which has been shown recently to be active against alginate, hyaluronic acid (HA) and poly-D-glucuronic acid at varying pH values (J. Biol. Chem. 289: 312 ( 2014)). The active site appears to have regions specific to binding these three sugars and an active catalytic site. Our objective is to understand how pH influences binding of a HA and if this involves structural changes in the protein. Docking of HA to a homology-based structure of Smlt1473 (based on the crystal structure of alginate lyase A1-III) was used to provide initial starting conformations for our molecular dynamics (MD) simulations. ProPKa was used to determine the ionization state of residues at our three simulated pHs (5, 7 and 9). MD simulations at neutral pH confirm the binding residues seen in experiment. However, at the acidic and basic condtions, the homology-based model of the binding pocket opens. At ph¼5, there is an increase in the exposure of HA-specific residues in the vicinity of W171 suggesting this exposure is the cause for Smlt1473 increased activity (J. Biol. Chem. 289: 312 ( 2014)). Therefore, Smlt1473's structure appears to strongly depend on pH and is used to control its activity toward different sugars.

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prone to aggregation. In this study, to overcome this issue and chemically regulate disulfide-coupled peptide and protein folding, a series of chemical reagents was examined in the refolding of hepcidin and prohepcidin, as a model peptide and protein, and their folding recoveries were estimated. Hepcidin consists of 25 amino acid residues and four intra-molecular disulfide bonds, which are absolutely required for its biological activity [2], not only for iron homeostasis, but also for anti-microbial activity. To investigate structurefunction relationships, hepcidin was chemically synthesized. However, the yield of synthesized hepcidin was quite low under the typical folding conditions. The major problem in the disulfide-coupled folding of hepcidin is that it undergoes aggregation during its folding reaction [3]. To solve this problem, several types of redox reagents and solvents were examined to improve the folding efficiency of hepcidin [3]. However, all of the reagents resulted in quite low yields for the disulfide-coupled folding of hepcidin. Therefore, to regulate the folding reaction of hepcidin and its precursor protein, we estimated the folding conditions, such as pH values, salt concentrations, and a variety of redox reagents. The results will be discussed in this paper.[1] Chakravarthi, S.; Jessop, C.E.; Bulleid, N.J. EMBO reports 2006, 7, 271-275. [2] Hocquellet, A.; le Senechal, C.; Garbay, B. Peptides 2012, 36, 303-307. [3] Zhang, J.; Diamond, S.; Arvedson, T.; Sasu, BJ.; Miranda, LP. Biopolymers 2010, 94, 257-264. Human cystatin C (HCC) is a cysteine protease inhibitor. This protein in pathological conditions, forms dimers via a ''domain swapping'' mechanism. HCC is also associated with two types of amyloid deposition diseases -hereditary amyloid angiopathy (related to the Leu68Gln mutation) and wild-type cystatin C co-precipitation. The aim of our studies was the characterisation of the self-assembling properties of native and mutated (at positions 57 or 68) forms of human cystatin C in solution. The structure, overall conformation and secondary structure changes in solution were studied by Fourier transformed infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), dynamic light scattering (DLS) and time resolved small angle scattering of synchrotron radiation (TR-SAXS). SAXS data for native and mutated HCC were subjected to analysis by using SVD and MCR-ALS methods as well as the low resolution structure determination. Besides the monomeric forms of human cystatin C, also dimers and higher oligomers were formed even after short (50-ms) exposure on synchrotron radiation. In addition we observed for first time, formation of domain swapped dimers of human cystatin C induced by irradiation. The spectroscopic studies confirmed conformational changes. Light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits...