No description available.

The 105aa lambda S protein is the prototype holin, S accumulates in the cytoplasmic membrane during late gene expression until, at a time programmed into its primary structure, it disrupts the membrane and allows the lambda lysozyme, R, to attack the cell wall. In this study, a zwitterionic detergent Empigen BB, was used to extract and purify the lambda holin S. In Empigen BB, CD analysis on S gave 54% alpha helical content, consistent with 3 TM domains, which has been reported by other in vivo studies. Empigen BB-purified S can be exchanged into a chaotropic solution by dialysis and reconstituted into preformed lipid vesicles for activity assays. When diluted to fluorescein-loaded suspensions of liposomes, different chaotrope-solubilized S alleles caused dye release reflective of their in vivo phenotypes. The problem was the low efficiency of delivery of S to the liposomes. Unfortunately, dye loaded liposomes are highly sensitive to any detergent, making it necessary to find other ways to solubilize S. GroEL, a chaperonin from E. coli, is responsible for folding and refolding globular proteins in vitro. It has also been reported that GroEL improves the ability of a membrane protein synthesized in vitro to insert post-translationally into liposomes. This work will investigate the behavior of GroEL towards membrane proteins. The first of two membrane proteins studied in this respect is Bacteriorhodopsin (BR), a membrane proton pump, from H. halibium. The second is the105aa S protein, a prototype holin from bacteriophage lambda. Holin and BR subjected to detergent removal in the presence of GroEL remained in solution, while in the control sample (without GroEL) S and BR precipitated. "GroELsolubilized" holin still retained its lesion forming activity and solubilized BR maintained its proton pumping ability, detected by using a liposome dye activity assay unique to each protein. This approach may be applicable to other systems requiring detergent- or chaotrope-free preparation of membrane proteins. Finally, these results suggest that GroEL may be involved in the insertion of integral membrane proteins into the lipid bilayer, a role heretofore unsuspected.

Elucidating the structure-function relationships of membrane proteins is critical for the design of therapeutic agents to treat disease and for understanding numerous cellular processes such as signal transduction and molecular or ion transport. Recent advances in the application of correlated single molecule imaging techniques have provided new insights into protein-protein and protein-membrane interactions. To demonstrate the potential of these approaches, we have used in situ atomic force microscopy and single molecule fluorescence microscopy to characterize the interactions between membrane receptors and their soluble ligands, examine the monomer-dimer equilibrium in a family of adhesion receptors, and elucidate protein-mediated membrane restructuring of a supported lipid bilayer. Building on these studies, we examined the CorA ion transporter protein. We demonstrated single molecule resolution of reconstituted CorA molecules in supported lipid bilayers using a correlated AFM-TIRF microscopy platform. This approach provided new insights into a purported mechanism of CorA activation that involved ion binding.

Elucidating the structure-function relationships of membrane proteins is critical for the design of therapeutic agents to treat disease and for understanding numerous cellular processes such as signal transduction and molecular or ion transport. Recent advances in the application of correlated single molecule imaging techniques have provided new insights into protein-protein and protein-membrane interactions. To demonstrate the potential of these approaches, we have used in situ atomic force microscopy and single molecule fluorescence microscopy to characterize the interactions between membrane receptors and their soluble ligands, examine the monomer-dimer equilibrium in a family of adhesion receptors, and elucidate protein-mediated membrane restructuring of a supported lipid bilayer. Building on these studies, we examined the CorA ion transporter protein. We demonstrated single molecule resolution of reconstituted CorA molecules in supported lipid bilayers using a correlated AFM-TIRF microscopy platform. This approach provided new insights into a purported mechanism of CorA activation that involved ion binding.

Microorganisms primarily acquire phosphorus (P) in the form of inorganic phosphate (PO4-3 or Pi) through expression of a suite of phosphate scavenging or phosphate transporter systems in response to limiting environmental phosphate. One such system is the Pit family of single protein Pi transport systems found in all domains. These vary in size from 300 to 800 amino acids (a.a.) in size. Previously, the pit gene of the soil bacterium Sinorhizobium meliloti, was found to encode a 334 a.a. Pi uptake system (KM 1-2µM) that is repressed in low Pi conditions. However, the S. meliloti pit gene is encoded in an operon and overlaps the coding sequence of a protein of unknown function, which was denoted as pap (pit-accessory protein). Using a conditional Pi-transport deficient mutant strain of S. meliloti, the effects of pap or pit mutations on Pit-mediated Pi uptake were studied by conducting growth experiments in minimal media (with Pi as the sole source of P) and Pi uptake experiments. Both pap and pit deletions resulted in a loss of growth and Pi uptake, which could be complemented by integration of the pap and pit genes into the deletion locus. Heterologous Pap-Pit systems from Bacteroides thetaiotaomicron and Shewanella oneidensis were found to have KM values (17 and 8.5 µM, respectively) similar to previously reported values of S. meliloti Pap-Pit. However, the Shewanella Pit protein was capable of transporting Pi in the absence of the cognate Pap protein, albeit with greatly reduced velocity at all measured concentrations. Pap-Pit orthologs were identified in ~2000 diverse prokaryotic proteomes using Pfam motifs of Pit (PHO4) and Pap (PhoU_div) protein domains. pap-pit operons were found in a third of all proteomes, and were predicted to be a co-transcribed operon in >95% of cases. This provided additional evidence that Pap is directly involved in Pit-mediated Pi uptake, and also that Pap-Pit systems have a significant role in microbial Pi uptake. Pap protein sequences and structures show striking similarities with that of PhoU, a protein of unknown function implicated as a modulator of the Pst uptake system. Pap and PhoU proteins share highly conserved putative metal-binding motifs (E/DXXXD) of which several Pap missense mutations were found to result in reduced Pi transport. This suggests that like PhoU, Pap may function as a modulator of Pi uptake by an interaction with its cognate transporter, Pit. However, the molecular mechanisms of PhoU and Pap proteins have yet to be defined. / Thesis / Master of Science (MSc) / Microbes acquire and assimilate phosphorus (P) in the form of inorganic phosphate (Pi) through a variety of mechanisms. Pit (Pi transporter) are a family of diverse transporters found in all kingdoms of life. Unlike other Pit systems, the Sinorhizobium meliloti pit gene is encoded in an operon with a protein of unknown function, denoted the pit-accessory protein (pap). Using S. meliloti Pap-Pit and orthologues from other bacteria as model systems, we demonstrate that Pap functions as a positive modulator of Pi uptake via Pit, as Pap is required for active uptake of Pi. Pap-Pit systems are found in 30% of all bacteria and archaea, and thus broadly distributed. Understanding the mechanism of Pap-Pit has biotechnological applications, as multiple Pap-Pit systems are present in phosphorus-accumulating bacteria utilized for waste-water treatment.

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The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

<p>ZMPSTE24 is a unique intramembrane zinc metalloprotease that plays critical roles in the lamin A maturation pathway. Lamin A comprises a dense network underlying the inner nuclear membrane that maintains the structural integrity and proper function of the nucleus. The precursor of lamin A, prelamin A, terminates with a CAAX motif, where “C” is cysteine, “A” is typically an aliphatic amino acid, and “X” is one of several different amino acids. Like all CAAX proteins, prelamin A undergoes a series of post-translational modifications, including farnesylation of the cysteine by farnesyltransferase, endoproteolysis of the AAX residues by ZMPSTE24 or possibly a related protease RCE1, and carboxyl methylation by isoprenylcysteine carboxyl methyltransferase (ICMT). After CAAX processing, an additional cleavage event by ZMPSTE24 occurs to remove 15 residues from the C-terminus, including the farnasylated and carboxyl methylated cysteine, releasing mature lamin A into the nucleoplasm. Mutations in the gene encoding ZMPSTE24 that impair proteolytic activity cause a set of progeroid diseases, including B-type mandibuloacral dysplasia (MAD-B) and restrictive dermopathy (RD). Recently, ZMPSTE24 mutations were also detected in patients with metabolic syndrome (MS) and nonalcoholic fatty liver disease (NAFLD). Patients with these diseases have shown defective prelamin A processing, leading to the accumulation of persistently farnesylated prelamin A in the nucleus. However, how this accumulation causes disease remains unclear. We demonstrated that both ZMPSTE24 and another known CAAX protease, RCE1 are both capable of mediating the C-terminal cleavage of prelamin A. RCE1 retains the capacity to cleave the AAX residues of prelamin A in progeroid diseases induced by inactive ZMPSTE24 mutants, therefore the disease molecule will be most likely farnesylated and methylated prelamin A. These factors suggest that the ability of ZMPSTE24 to perform the upstream cleavage determines the accumulation level of uncleaved prelamin A in progeroid diseases. However, there was no available assay that could quantitatively demonstrate the <i>in vitro</i> upstream cleavage activity of ZMPSTE24. Therefore, we first developed a FRET-based assay that was able to precisely quantify the upstream cleavage activity of Ste24, the yeast homolog of ZMPSTE24. The 33-mer analog of <b>a</b>-factor, Ste24 natural substrate, has a 2-aminobenzoic acid (Abz) fluorophore at the N-terminus and a dinitophenol (Dnp) quencher located on the either side of the proposed cleavage site. After cleavage, quantification of the fluorescence from the dequenced peptide enabled us to continuously monitor the upstream cleavage activity of Ste24. We then utilized this FRET-based assay to examine the upstream cleavage activity of wild-type ZMPSTE24 and its disease mutants. We demonstrated that the <b>a</b>-factor analog FRET substrate could be recognized and cleaved at the predicted position by ZMPSTE24. Disease variants were examined using this assay and the results revealed reduced upstream cleavage activity. Moreover, blocking ubiquitylation restored catalytic activity of some ZMPSTE24 disease variants, suggesting that diminished activity of these mutants is due to protein instability. Limited trypsin digestion results also indicated that some variants may not be properly folded, as compared to wild-type. The crystal structure of ZMPSTE24 revealed that seven transmembrane helices of ZMPSTE24 surround a large intramembrane chamber. The HEXXH zinc metalloprotease motif faces inward to cap the top of the chamber inside which proteolysis is proposed to occur. The four side portals apparent in the structure may provide substrate entry and exit routes. Based on structural considerations, besides interfering with structural integrity, these disease mutations may decrease ZMPSTE24 activity by preventing substrate binding in the active site or occluding substrate entry into or exit from the enzyme chamber. Using a yeast <b>a</b>-factor sequence-based photoactive analog containing benzophenone in the farnesyl portion, which can be well processed by ZMPSTE24, we have shown that certain disease mutants may affect farnesyl binding of the C-terminal cleavage substrate. We also designed several double cysteine mutants near portal 1 in Cys-less background of Ste24. We then utilized bismaleimide sulfhydryl-to-sulfhydryl crosslinkers to block the portal opening. Some of the crosslinked double mutants showed reduced AAX cleavage activity, suggesting the portal 1 may be important for the C-terminal cleavage. Together, these data will clarify how the enzyme functions and also provide further insights into progeroid diseases.</p>