John E. Wampler

Occurrence and role ofcis peptide bonds in protein structures

It has been widely assumed that the occurrence of cis peptide bonds in proteins is quite rare due to unfavorable contacts between adjacent amino acid residues in this isomeric form. To investigate this assumption, the Brookhaven Protein Data Bank was examined for the occurrences of cis peptide bonds. Out of 31,005 amide bonds, only 17, or 0.05%, are cis, while 99 of the 1534 imide bonds (X-Pro), or 6.5%, are cis. These figures are considerably less than the distribution predicted on the basis of the potential energy difference between the cis and trans isomeric forms and experimental data on small peptides. It is not known whether the lower than expected occurrence of cis peptide bonds arises from constraints imposed by the protein environment, or from assumptions made in the solution of the X-ray crystal structures. However, when the occurrence of cis bonds in the data base is examined relative to the resolution of the structures, the number of cis bonds increases with increasing resolution. The distributions seen for these peptide omega bonds in the data base are not the same shape as the distributions predicted from simple potential energy barriers. They are sharper in the main, but they are also broader at the base with significant numbers of nonplanar peptide bonds. Cis peptide bonds are found primarily in bends and turns and, in the case of cis imide bonds (X-PRO), this correlation is so high that it suggests a specific role for cis imide groups in such structures.

BiGGER: A new (soft) docking algorithm for predicting protein interactions

A new computationally efficient and automated “soft docking” algorithm is described to assist the prediction of the mode of binding between two proteins, using the three‐dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6‐dimensional binding spaces of both molecules is systematically searched. A population of candidate protein‐protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein‐protein complexes of known crystallographic structures. In 22 out of 25 protein‐protein complexes tested, near‐native docked geometries were found with Cα RMS deviations ≤ 4.0 Å from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed. Proteins 2000;39:372–384.

An On-Line Spectrofluorimeter System for Rapid Collection of Absolute Luminescence Spectra:

A spectrofluorimeter system has been developed which uses an on-line general purpose digital computer to both collect and correct fluorescence and luminescence spectra; no servosystems or complex analog circuitry are employed. Spectra are represented by a series of up to 500 individual data points, each of which is, in turn, the average of 16 samplings of the signal photomultiplier. The signal produced by a reference photomultiplier is also collected and used to make corrections appropriate to the type of spectra being collected. Wavelength drive is accomplished with a reversible stepping motor driven by commands from the computer; thus the wavelength position can be automatically and precisely controlled by the computer without recourse to cumbersome mechanical components. Corrections to collected spectra are made in real time on the basis of correction factors derived from fluorescence standards or a standard lamp. The complete system can collect a full spectrum (500 points) in 8 sec, average the results of any number of automatically repeated scans, and furnish the operator with both corrected and uncorrected spectra on either a wavelength or a wavenumber scale. In addition, the data can be retained on punched paper tape or presented graphically in a format acceptable for publication. Details of the system are presented with examples of its performance.

An automated dual-wavelength spectrophotometer optimized for phytochrome assay

ABSTRACT A microcomputer-controlled dual-wavelength spectrophotometer suitable for automated phytochrome assay is described. The optomechanical unit provides for sequential irradiation of the sample by the two measuring wavelengths with intervening dark intervals and for actinic irradiation to inter-convert phytochrome between its two forms. Photomultiplier current is amplified, converted to a digital value and transferred into the computer using a custom-designed IEEE-488 bus interface. The microcomputer calculates mathematically both absorbance and absorbance difference values with dynamic correction for photomultiplier dark current. In addition, the computer controls the operating parameters of the spectrophotometer via a separate interface. These parameters include control of the durations of measuring and actinic irradiation intervals and their sequence. Because the data are processed solely within the microcomputer, except for the initial linear amplification, any output desired can be obtained, inc...

Earthworm bioluminescence: Comparative physiology and biochemistry

Abstract 1. 1. The comparative physiology and biochemistry of twelve species of bioluminescent earthworms from six genera (Diplocardia, Diplotrema, Fletcherodrilus, Octochaetus, Pontodrilus and Spenceriella) from the U.S., Australia and New Zealand indicated considerable similarity in the mechanism of bioluminescence. 2. 2. With the exception of Pontodrilus bermudensis where the fluid is exuded from the mouth, all species exude bioluminescent coelomic fluid from their dorsal pores. 3. 3. Microscopic observation, isolation of active particulate fractions from seven species and comparison to the previous study of D. longa (Rudie & Wampler, 1978) suggests that the site of luminescence is the free chloragogen cell. 4. 4. The emission spectra (eight species) were broad, unimodal with maxima ranging from 500 nm to greater than 570 nm. 5. 5. The spectral and biochemical data supports the hypothesis that earthworm bioluminescence involves the reaction of hydrogen peroxide with N- isovaleryl-3-aminopropanal or its close analog, and that spectral distribution is determined by the luciferase or other luciferase-association components.

Similarities in the bioluminescence from the Pennatulacea.

Abstract 1. The in vivo bioluminescence spectra of six species of cnidarians from the subclass Alcyonaria, order Pennatulacea, have been examined. They are all strikingly similar having as a predominant feature a narrow green emission peak with a maximum at 19 640 cm−1 (509 nm). Each of these spectra is structured with a shoulder in the region of 18 500 cm−1 (540 nm) and in some cases another shoulder in the 21 000-cm−1 (475-nm) region. 2. A protein bound green-fluorescent chromophore has been isolated from each of these animals. The fluorescence spectra of these chromophores are identical. Their peak position, 19 640 cm−1 (509 nm), and narrow structured character strongly suggest that they represent the emitter responsible for the corresponding parts of the bioluminescence spectra. 3. The data indicate that in each of these Pennatulids, the overall bioluminescence spectrum can be explained as the combination of two emitting species, the fluorescence of the green-fluorescent, protein-bound chromophore and the broad blue fluorescence typical of the in vitro reactions.

SPECTRAL ANALYSIS OF BIOLUMINESCENCE OF PANELLUS STYPTICUS

Corrected in vivo bioluminescence emission spectra have been obtained from monokaryotic and dikaryotic cultures and mature fruiting bodies of Pannellus stypticus. The wavelength of maximum bioluminescence was found at 525 nm. The bioluminescence emission was shifted to longer wavelengths, with a maximum at 528 nm, in basidiocarps that were deeply pigmented. The wavelengths of maximum bioluminescence of a number of fungi reported in the literature were determined to be from uncorrected spectra that legitimately cannot be compared with each other.

Chapter 14 Quantitative Fluorescence Microscopy Using Photomultiplier Tubes and Imaging Detectors

Publisher Summary This chapter describes the quantitative fluorescence microscopy using photomultiplier tubes and imaging detectors. The most typical geometry for fluorescence microscopy is the epi-illumination geometry, where the exciting light is delivered to the sample through the objective lens. The main role of lenses in the illumination light path is to deliver the light to the limiting aperture and control its uniformity. Light rays emitted from the source are collected by a focusable lens or group of lenses called the collector lens. In the epifluorescence microscope, the objective functions as a condenser. This relationship yields a gain in brightness as a function of the fourth power of the numerical aperture of the objective. The choice of objective lens is critical for quantitative fluorescence microscopy. Each objective used must have high transmission efficiency and contribute no fluorescence from its internal parts. In most applications there are two additional criteria, high light collection efficiency and good resolution. The returning fluorescence signal in a conventional epifluorescence microscope must pass the beam splitter and any intermediated optics used by the manufacturer to control the internal light path.

Spectral characteristics of the bioluminescence induced in the marine fish,Porichthys notatus, byCypridina (ostracod) luciferin

SummarySpecimens ofPorichthys notatus, which are naturally luminous along the coast of California, are non-luminous in Puget Sound. However, luminescence capability may be induced in the adult Puget SoundPorichthys by the administration of purifiedCypridina (ostracod) luciferin, syntheticCypridina luciferin, orCypridina organisms. The bioluminescence emission spectra produced by the Puget Sound fish following induction is similar, if not identical, to that of the naturally luminousPorichthys notatus from California waters (maxima: 485 and 507 nm).

Earthworm bioluminescence: Characterization of the luminescent cell from Diplocardia longa

Abstract 1. 1. Diplocardia longa bioluminescence originates from discrete subcellular loci within free chloragogen cells suspended in the coelomic fluid. 2. 2. The free chloragogen cells are replete with granular particles of two or three types but contain little other subcellular structure. 3. 3. Chloragogen tissue from the vicinity of the dorsal blood vessel also contains components of the luminescent system. 4. 4. Attempts to isolate the subcellular component containing the packaged luminescence system were partially successful in that a low density, active fraction was obtained, suggesting association of the system with membrane or other lipid.