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P C Jones

Publications and source records attributed to P C Jones.

At least 19 recordsLinked to original sources
PubMed2000

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase.

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.

Adenosine Triphosphate↗
PubMed2000

Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase.

Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases. The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling. In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer. Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer. The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis. Rather, the results support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer. Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E. coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases. A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution.

Adenosine Triphosphate↗
PubMed1999

Structure of the subunit c oligomer in the F1Fo ATP synthase: model derived from solution structure of the monomer and cross-linking in the native enzyme.

The structure of the subunit c oligomer of the H+-transporting ATP synthase of Escherichia coli has been modeled by molecular dynamics and energy minimization calculations from the solution structure of monomeric subunit c and 21 intersubunit distance constraints derived from cross-linking of subunits. Subunit c folds in a hairpin-like structure with two transmembrane helices. In the c12 oligomer model, the subunits pack to form a compact hollow cylinder with an outer diameter of 55-60 A and an inner space with a minimal diameter of 11-12 A. Phospholipids are presumed to pack in the inner space in the native membrane. The transmembrane helices pack in two concentric rings with helix 1 inside and helix 2 outside. The calculations strongly favor this structure versus a model with helix 2 inside and helix 1 outside. Asp-61, the H+-transporting residue, packs toward the center of the four transmembrane helices of two interacting subunits. From this position at the front face of one subunit, the Asp-61 carboxylate lies proximal to side chains of Ala-24, Ile-28, and Ala-62, projecting from the back face of a second subunit. These interactions were predicted from previous mutational analyses. The packing supports the suggestion that a c-c dimer is the functional unit. The positioning of the Asp-61 carboxyl in the center of the interacting transmembrane helices, rather than at the periphery of the cylinder, has important implications regarding possible mechanisms of H+-transport-driven rotation of the c oligomer during ATP synthesis.

Alanine↗
PubMed1999

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.

Amino Acid Sequence↗
PubMed1998

Genetic fusions of subunit c in the F0 sector of H+-transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis.

The multicopy c subunit of the H+-transporting ATP synthase of Escherichia coli folds through the transmembrane F0 sector as a hairpin of two hydrophobic alpha-helices with the proton-translocating aspartyl-61 side chain centered in the second transmembrane helix. The number of subunits c in the F0 complex, which is thought to determine the H+-pumping/ATP stoichiometry, was previously not determined with exactness but thought to range from 9-12. The studies described here indicate that the exact number is 12. Based upon the precedent of the subunit c in vacuolar-type ATPases, which are composed of four transmembrane helices and seem to have evolved by gene duplication of an F0-type progenitor gene, we constructed genetically fused dimers and trimers of E. coli subunit c. Both the dimeric and trimeric forms proved to be functional. These results indicate that the total number of subunit c in F0 should be a multiple of 2 and 3. Based upon a previous study in which the oligomeric organization of c subunits in F0 was determined by cross-linking of Cys-substituted subunits (Jones, P. C. , Jiang, W., and Fillingame, R. H. (1998) J. Biol. Chem. 273, 17178-17185), we introduced Cys into the first and last transmembrane helices of subunit c monomers, dimers, and trimers and attempted to generate cross-linked products by oxidation with Cu(II)-(1,10-phenanthroline)2. Double Cys substitutions at two sets of positions gave rise to extensive cross-linked multimers. Multimers of the monomer that extended up to the position of c12 were correlated and calibrated with distinct cross-linked species of the appropriate doubly Cys-substituted dimers (i.e. c2, c4, . c12) and doubly Cys-substituted trimers (i.e. c3, c6, c9, c12). The results show that there are 12 copies of subunit c per F0 in E. coli, the exact number having both mechanistic and structural significance.

Artificial Gene Fusion↗
PubMed1998

Arrangement of the multicopy H+-translocating subunit c in the membrane sector of the Escherichia coli F1F0 ATP synthase.

The multicopy subunit c of the H+-transporting F1F0 ATP synthase of Escherichia coli is thought to fold across the membrane as a hairpin of two hydrophobic alpha-helices. The conserved Asp61, centered in the second transmembrane helix, is essential for H+ transport. In this study, we have made sequential Cys substitutions across both transmembrane helices and used disulfide cross-link formation to determine the oligomeric arrangement of the c subunits. Cross-link formation between single Cys substitutions in helix 1 provided initial limitations on how the subunits could be arranged. Double Cys substitutions at positions 14/16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the formation of cross-linked multimers upon oxidation. Double Cys substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and 20/66, respectively, also formed cross-linked multimers. These results indicate that at least 10 and probably 12 subunits c interact in a front-to-back fashion to form a ring-like arrangement in F0. Helix 1 packs at the interior and helix 2 at the periphery of the ring. The model indicates that the Asp61 carboxylate is centered between the helical faces of adjacent subunit c at the center of a four-helix bundle.

Adenosine Triphosphate↗
PubMed1998

Subunit organization and structure in the F0 sector of Escherichia coli F1F0 ATP synthase.

In this review, we summarize recent work from our laboratory which establishes the topology and nearest neighbor organization of subunits in the F0 sector of the H+ transporting ATP synthase of Escherichia coli. The E. coli F0 sector is composed of three subunits in an a1b2c12 stoichiometric ratio. Crosslinking experiments with genetically introduced Cys establish a ring-like organization of the 12 c subunits with subunits a and b lying to the outside of the ring. The results are interpreted using an atomic resolution structural model of monomeric subunit c in a chloroform-methanol-water (4:4:1, v/v/v) solution, derived by heteronuclear NMR (M.E. Girvin, F. Abildgaard, V. Rastogi, J. Markley, R.H. Fillingame, in press). The crosslinking results validate many predictions of the structural model and confirm a front-to-back-type packing of two subunit c into a functional dimer, as was first predicted from genetic studies. Aspartyl-61, the proton translocating residue, lies at the center of the four transmembrane helices of the functional dimer, rather than at the periphery of the subunit c ring. Subunit a is shown to fold with five transmembrane helices, and a functionally important interaction of transmembrane helix-4 with transmembrane helix-2 of subunit c is established. The single transmembrane helices of the two subunit b dimerize in the membrane. The structure of the transmembrane segment of subunit b is predicted from the NMR structure of the monomeric peptide.

Dimerization↗
PubMed1998

Electronic communication and collaboration in a health care practice.

Using cognitive evaluation techniques, this study examines the effects of an electronic patient record and electronic mail on the interactions of health care providers. We find that the least structured communication methods are also the most heavily used: face-to-face, telephone, and electronic mail. Positive benefits of electronically-mediated interactions include improving communication, collaboration, and access to information to support decision-making. Negative factors include the potential for overloading clinicians with unwanted or unnecessary communications.

Communication↗
PubMed1998

Telematics in the neonatal ICU and beyond: improving care, communication and information sharing.

In October of 1998, the Beth Israel-Deaconess was awarded one of 19 contracts from the National Library of Medicine (NLM) to develop, implement and test a telemedicine application to support the care of Very Low Birth Weight Infants. This project is the only one to focus on the care of newborns. We believe that this project will provide a new national approach to managing the care of high-risk newborns by leveraging evolving communication technology.

Computer Security↗
PubMed1998

Nationwide telecare for diabetics: a pilot implementation of the HOLON architecture.

This paper presents results from a demonstration project of nationwide exchange of health data for the home care of diabetic patients. A consortium of industry, academic, and health care partners has developed reusable middleware components integrated using the HOLON architecture. Engineering approaches for multi-organization systems development, lessons learned in developing layered object-oriented systems, security and confidentiality considerations, and functionality for nationwide telemedicine applications are discussed.

Computer Communication Networks↗
PubMed1998

Baby CareLink: development and implementation of a WWW-based system for neonatal home telemedicine.

Baby CareLink is a multifaceted telemedicine application designed to provide individualized information and support to families of Very Low Birth Weight infants. We believe that this innovative use of WWW and telemedicine technologies will improve family satisfaction and clinical care. In conjunction with improvements in family involvement, discharge planning, education, and follow-up enabled by other CareLink components, this system may allow infants to transition home even earlier in their hospital stay and thereby provide a clear cost savings. This paper discusses the CareLink architecture and lessons learned in implementing a telemedicine link with families at home from an in-hospital clinical unit.

Computer Security↗
PubMed1997

Engineering protein mechanics: inhibition of concerted motions of the cellular retinol binding protein by site-directed mutagenesis.

Recently we reported on the dynamic properties of the cellular retinol binding protein, a member of the fatty acid binding protein family. A few conserved glycines were identified as important for producing the conformational changes necessary for the uptake and release of retinol. Here, we describe a multidisciplinary analysis of a genetically engineered mutation of one of these glycines (Gly67), designed to inhibit an observed hinge bending motion. The correctly folded mutant protein is unable to bind retinol. Analysis of the molecular dynamics simulations of the mutant and wild type protein using the essential dynamics method shows that the mutation indeed inhibits the hinge bending motions which are important for retinol binding.

Binding Sites↗
PubMed1997

Telematics in the neonatal ICU and beyond: improving care for high-risk newborns and their families.

The Beth Israel-Deaconess has recently been awarded one of 19 contracts from the National Library of Medicine (NLM) to develop, implement and test a telemedicine application to support the care of Very Low Birth Weight Infants. This project is the only one to focus on the care of newborns. We believe that this project will provide a new national approach to managing the care of high-risk newborns by leveraging evolving communication technology.

Computer Security↗
PubMed1995

The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase.

The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.

Animals↗
PubMed1995

Membrane insertion and assembly of ductin: a polytopic channel with dual orientations.

Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)-ATPase (V-ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.

Amino Acid Sequence↗
PubMed1994

Functional properties of a hybrid vacuolar H(+)-ATPase in Saccharomyces cells expressing the Nephrops 16-kDa proteolipid.

The hydrophobic 16-kDa polypeptide which forms gap-junction-like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H(+)-ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H(+)-ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild-type V-ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction. To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni(2+)-binding support, six C-terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H(+)-ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane-bound Vo sector of the ATPase was isolated by the affinity-chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.

Adenosine Triphosphate↗