PubMed Health⌕ Search

Biomedical subjects

S L Helgerson

Publications and source records attributed to S L Helgerson.

10 recordsLinked to original sources
PubMed1992

NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.

Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.

Amino Acid Sequence↗
PubMed1989

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer.

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for Förster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1989

Isoelectric focusing studies of bacteriorhodopsin.

Purified bacteriorhodopsin (BR) samples show a minimum of four isoelectric forms in immobilized pH gradient isoelectric focusing gels. The bands occur as doublets with isoelectric points (pI) centered at 5.20 (principal species) and 5.60. In typical preparations additional bands may be observed at 4.90, 5.07 and 5.50. Purple membrane (PM) was proteolyzed with papain to calibrate the pI shift produced by changing the number of charges on the protein. Asp-242 is removed during the first cleavage between residues 239 and 240 resulting in the loss of a single negative charge and a shift of the principal doublet by +0.35 pH units to pI 5.55. The second papain cleavage occurs between residues 231 and 232 which removes Glu-232, -234 and -237 and shifts the pI by +0.60 pH units to pI 6.10. The +0.60 pH shift upon the second papain cleavage is consistent with the loss of two negative charges and is supported by prior evidence that at least one of the three glutamate residues lost during the second proteolysis step is protonated and neutral in the intact protein. The native and proteolyzed products of BR retain the characteristic 550 nm absorption maxima for solubilized BR. A model for the structural origin of the pI heterogeneity of BR species in proteolyzed PM is presented.

Bacteriorhodopsins↗
PubMed1987

Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a platelet cell-surface receptor for fibrinogen and fibronectin. A carboxyl-terminal decapeptide of the fibrinogen gamma-chain (Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val LGGAKQAGDV] and a tetrapeptide (Arg-Gly-Asp-Ser (RGDS] from the fibrinogen alpha-chain and the fibronectin cell-binding domain appear to mediate the binding of these ligands to GP IIb-IIIa. The present study was designed to examine the effects of these and related peptides on the structure of purified platelet GP IIb-IIIa. Treatment of GP IIb-IIIa with various synthetic peptides affected the glycoprotein so that GP IIb alpha became a substrate for hydrolysis by thrombin. The order of potency of these peptides was as follows: RGDS greater than LGGAKQAGDV greater than KGDS greater than RGES. This is the same order of potency in which these peptides inhibit fibrinogen binding to platelets. This effect was time-, temperature-, and concentration-dependent; RGDS induced a half-maximal effect at approximately 60 microM. In addition, RGDS, but not RGES, decreased the intensity of the intrinsic protein fluorescence of GP IIb-IIIa. Finally, the decapeptide or RGDS decreased the sedimentation coefficient of GP IIb-IIIa from 8.5 to 7.7 or 7.4 S, respectively, whereas RGES had a minimal effect. This decrease was accompanied by an increase in the Stoke's radius from 74 to 82 A with RGDS or 85 A with the decapeptide, indicating a peptide-induced unfolding of the GP IIb-IIIa complex. This change in conformation may be related to changes in the distribution and function of GP IIb-IIIa on the platelet surface that occur when adhesive proteins or peptides from the GP IIb-IIIa binding domains of these proteins bind to GP IIb-IIIa.

Binding Sites↗
PubMed1976

An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli.

Colicin El and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli. It has been shown elsewhere that this fluorescence increase correlates well with de-energization. Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm. These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level. The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport; (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing eutger cinpetition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition. (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization. However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization. Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly. Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe. The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.

1-Naphthylamine↗
PubMed1976

Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry.

The stationary fluorescence polarization and the differential phase delay of the polarized components of the fluorescence of 1-phenylnaphthylamine in Escherichia coli suspensions were measured before and after addition of colicin E1. Both sets of measurements register an increase in the rotational relaxation time of the fluorescent probe when colicin is present. These increases are absent in an E. coli mutant tolerant to colicin E1. The physical interpretation of the changes demands separate estimation of the fraction f2 of the emitting fluorophores that change their properties upon colicin addition and of the rotational relaxation time p2 of this fraction, following the colicin-induced changes. By themselves, the steady state polarizaiton observations permit only the conclusion that f2 must be in the range of 1-0.06 and the change in p2/pi between 1.5 and a value larger than 10. Combination of the data of stationary polarization with those of differential phase fluorometry results in an important reduction in the uncertainty:f2 must be in the range 1-0.33 and the change in p2/pi in the range 1.5-2.5.

Binding Sites↗
PubMed1976

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1.

It is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes. In intact E. coli ML 308-225 cells the inhibition of [14C]-PROLINE ACtive transport by FCCP increases with uncoupler concentration from approximately 20% at 2 muM to approximately 100% at 5 muM. The increase in the rotational relaxation time (rho) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)1 and 8-anilino-1-naphthalene-sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in rho value of ANS show the same dependence on FCCP concentration with saturation at 0.3 muM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5muM FCCP...

Biological Transport, Active↗