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1.
J Phys Chem A ; 117(29): 6311-22, 2013 Jul 25.
Article in English | MEDLINE | ID: mdl-23566310

ABSTRACT

Vibrational sum-frequency generation (VSFG) spectra of the amide-I band of proteins can give detailed insight into biomolecular processes near membranes. However, interpreting these spectra in terms of the conformation and orientation of a protein can be difficult, especially in the case of complex proteins. Here we present a formalism to calculate the amide-I infrared (IR), Raman, and VSFG spectra based on the protein conformation and orientation distribution. Based on the protein conformation, we set up the amide-I exciton Hamiltonian for the backbone amide modes that generate the linear and nonlinear spectroscopic responses. In this Hamiltonian, we distinguish between nearest-neighbor and non-nearest-neighbor vibrational couplings. To determine nearest-neighbor couplings we use an ab initio 6-31G+(d) B3LYP-calculated map of the coupling as a function of the dihedral angles. The other couplings are estimated using the transition-dipole coupling model. The local-mode frequencies of hydrogen-bonded peptide bonds and of peptide bonds to proline residues are red-shifted. To obtain realistic hydrogen-bond shifts we perform a molecular dynamics simulation in which the protein is solvated by water. As a first application, we measure and calculate the amide-I IR, Raman, and VSFG spectra of cholera toxin B subunit docked to a model cell membrane. To deduce the orientation of the protein with respect to the membrane from the VSFG spectra, we compare the experimental and calculated spectral shapes of single-polarization results, rather than comparing the relative amplitudes of VSFG spectra recorded for different polarization conditions for infrared, visible, and sum-frequency light. We find that the intrinsic uncertainty in the interfacial refractive index--essential to determine the overall amplitude of the VSFG spectra--prohibits a meaningful comparison of the intensities of the different polarization combinations. In contrast, the spectral shape of most of the VSFG spectra is independent of the details of the interfacial refractive index and provides a reliable way of determining molecular interfacial orientation. Specifically, we find that the symmetry axis of the cholera toxin B subunit is oriented at an angle of 6° ± 17° relative to the surface normal of the lipid monolayer, in agreement with 5-fold binding between the toxin's five subunits and the receptor lipids in the membrane.


Subject(s)
Amides/chemistry , Cholera Toxin/chemistry , Models, Molecular , Biomimetic Materials/chemistry , Hydrogen Bonding , Membranes, Artificial , Protein Conformation , Surface Properties
2.
Infect Immun ; 45(1): 290-2, 1984 Jul.
Article in English | MEDLINE | ID: mdl-6329958

ABSTRACT

Genes encoding the thermostable direct or Kanagawa phenomenon hemolysin of Vibrio parahaemolyticus were cloned in Escherichia coli. DNA hybridization experiments with the cloned genes showed that none of the five Kanagawa phenomenon-negative environmental isolates tested possessed DNA sequences homologous to the probe.


Subject(s)
Cloning, Molecular , Genes, Bacterial , Genes , Hemolysin Proteins/genetics , Vibrio parahaemolyticus/genetics , Base Sequence , DNA Restriction Enzymes , Hot Temperature , Immunodiffusion , Nucleic Acid Hybridization , Species Specificity
3.
Nucleic Acids Res ; 9(9): 2121-39, 1981 May 11.
Article in English | MEDLINE | ID: mdl-6272226

ABSTRACT

Fourteen different DNA fragments containing Escherichia coli tRNA genes have been cloned using the vector pBR322. We report the methods of cloning, the identification of specific tRNA genes, and the presence or absence of rRNA genes on these cloned DNA fragments. In particular, one chimeric plasmid contains a 17.0 kilobase pair EcoRI fragment bearing tRNA(Leu 1) sequences. Using nucleotide sequence analysis we have identified a cluster of three tandem tRNA(Leu 1) genes separated by intergenic spacers of 27 and 34 base pairs, respectively. The nucleotide sequence upstream of the first gene contains a transcription promoter site. A G-C rich sequence (5'-CGCCTCC-3') found between the Pribnow box and initiation site is very similar to the corresponding sequence found in other genes which are under stringent control.


Subject(s)
Escherichia coli/genetics , Operon , RNA, Transfer, Amino Acyl/genetics , Base Sequence , Cloning, Molecular/methods , DNA/genetics , DNA Restriction Enzymes , Deoxyribonuclease EcoRI , Nucleic Acid Hybridization , Plasmids , Transcription, Genetic
4.
J Bacteriol ; 144(3): 1083-93, 1980 Dec.
Article in English | MEDLINE | ID: mdl-6160141

ABSTRACT

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.


Subject(s)
Chromosomes, Bacterial , Escherichia coli/genetics , Genes , RNA, Bacterial/genetics , RNA, Transfer/genetics , Base Sequence , Chromosome Mapping , DNA Restriction Enzymes , Nucleic Acid Hybridization , RNA, Ribosomal/genetics
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