Interaction of small G proteins with photoexcited rhodopsin

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

Compartments of protein metabolism in the developing brain.

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins.     

How to measure and predict the molar absorption coefficient of a protein.

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.     

Use of stable isotopes to assess protein and amino acid metabolism in children and adolescents: a brief review

As protein accretion is a prerequisite for growth, studying the mechanisms by which nutrients and hormones promote protein gain is of the utmost relevance to paediatric endocrinology. Tracers are ideally suited for the assessment of protein and amino acid kinetics in vivo, as they provide an estimate of synthesis and turnover. Current tracer approaches in children and adolescents utilize stable isotopes, 'heavier' forms of elements that have one or several extra neutrons in the nucleus. Such isotopes are already present at low, but significant, levels in all tissues and foodstuffs, are not radioactive and are devoid of any known side-effects when present in small amounts. L-[1-(13)C] labelled leucine, given as a 4- to 6-h intravenous infusion, has become the method of choice to assess whole-body protein kinetics. After infusion, any 13C-leucine that is oxidized appears in the breath as 13CO2, whereas the remainder is incorporated into body proteins through protein synthesis. The isotope enrichments are determined by isotope ratio mass spectrometry and gas chromatography mass spectrometry, and absolute rates of whole-body protein synthesis, oxidation, and breakdown can be extrapolated. This approach has been used extensively to investigate the regulation of protein kinetics by nutrients and by hormones. Attempts have also been made to measure amino acid/protein metabolism in selected body compartments, and to measure the kinetics of specific tissue proteins, for example, muscle, gut, or plasma proteins.     

High sensitive protein detection by hollow fiber membrane interface based protein enrichment and in situ fluorescence derivatization

A novel device, composed of a syringe pump for sample loading, a hydrophilic hollow fiber membrane interface for protein concentration and small molecules removal, and a centrifugation tube for buffer exchange, was designed for protein preconcentration and in situ fluorescence derivatization. With the outlet of the interface blocked, denatured proteins were continually introduced. Restricted by the membrane with the molecular weight cutoff (MWCO) of 3000 Da, proteins were concentrated within the membrane. However, denaturant and other small molecules, which might affect the further fluorescence derivatization, were driven out of the membrane. Then, the membrane with proteins restricted inside was directly put into the fluorescence derivatization buffer. Here, the water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide of 3H-indolium,1-(5-carboxypentyl)-2-[3-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-,monopotassium salt (sw-cy3-NHS), synthesized in our lab, was used for protein labeling. By such a method, the detection sensitivity of bovine serum albumin (BSA) was improved by nearly 200 folds, compared to that obtained by direct in-solution derivatization. Through the derivatization of a fraction of E. coli protein separated by reversed phase HPLC, proteins with low concentration were efficiently labeled, which indicated the potential merit of the developed method for the high sensitive detection of low abundance proteins.     

Purification of riboflavin-binding proteins from bovine plasma and discovery of a pregnancy-specific riboflavin-binding protein.

Riboflavin-binding proteins have been purified from bovine plasma using flavinyl agarose beads. At least three major protein bands, migrating in regions assigned to the beta- and gamma-globulins of plasma, are observed by cellulose acetate electrophoresis. These proteins coelute from a calibrated Sephadex G-100 column in the volume corresponding to a molecular weight of approximately 150,000; a small amount of another riboflavin-binding protein (molecular weight approximately 37,000) is also present. Polyacrylamide gel electrophoresis of the proteins, with detection by autoradiography of those having tightly bound [2-14C]riboflavin, reveals one protein band which is present only in preparations from pregnant cows. This protein has been purified to apparent homogeneity by storing the mixture of riboflavin-binding proteins at 8 degrees C for 3 weeks, which precipitates the other, less stable proteins. Hence, bovine plasma, like that of the laying hen, contains a number of riboflavin-binding proteins, one of which correlates with pregnancy.     

Tissue porphyrin pattern determination by high-speed high-performance liquid chromatography

A double-label two-dimensional electrophoresis procedure has been developed which is specifically designed for the comparison of serum or plasma proteins in two different samples. Proteins are labeled by reductive methylation with [14C]- or [3H] formaldehyde. The procedure is economical because small quantities of relatively inexpensive isotopes are used and it is at least as sensitive as silver staining in detecting proteins. A fourfold increase in the sensitivity of autoradiography over existing methods was obtained by performing autoradiography before processing the gel for fluorography. A spot in the electrophoretic gel that contains 17-28 ng of labeled protein is detectable. This corresponds to proteins present in serum at a concentration of 5-10 micrograms/ml. Even greater sensitivity can be achieved, at greater expense, by increasing the quantities of the radioisotopes in the labeling reaction. The particular value of the double label approach is that complex mixtures from two different sources are resolved together thus eliminating the possibility of differences arising from the resolving procedure itself. The procedure was applied to a mixture of serum and plasma from a single subject and a number of qualitative and quantitative differences were observed.     

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa^1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol ³ . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography^4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples ⁶. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.     

Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins

A new algorithm, called convex constraint analysis, has been developed to deduce the chiral contribution of the common secondary structures directly from experimental CD curves of a large number of proteins. The analysis is based on CD data reported by Yang, J.T., Wu, C.-S.C. and Martinez, H.M. [Methods Enzymol., 130, 208-269 (1986)]. Application of the decomposition algorithm for simulated protein data sets resulted in component spectra [B (lambda, i)] identical to the originals and weights [C (i, k)] with excellent Pearson correlation coefficients (R) [Chang, C.T., Wu, C.-S.C. and Yang, J.T. (1978) Anal. Biochem., 91, 12-31]. Test runs were performed on sets of simulated protein spectra created by the Monte Carlo technique using poly-L-lysine-based pure component spectra. The significant correlational coefficients (R greater than 0.9) demonstrated the high power of the algorithm. The algorithm, applied to globular protein data, independent of X-ray data, revealed that the CD spectrum of a given protein is composed of at least four independent sources of chirality. Three of the computed component curves show remarkable resemblance to the CD spectra of known protein secondary structures. This approach yields a significant improvement in secondary structural evaluations when compared with previous methods, as compared with X-ray data, and yields a realistic set of pure component spectra. The new method is a useful tool not only in analyzing CD spectra of globular proteins but also has the potential for the analysis of integral membrane proteins.     

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].     

Identification of 5-Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex

5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.     

Methylation of Ribosomal Proteins in Escherichia coli

Escherichia coli was grown in a medium containing [1-(14)C]methionine and [methyl-(3)H]methionine, and the (3)H/(14)C ratio was determined for each of the ribosomal proteins derived from the 70S ribosome. Evidence indicates that six proteins from the 50S subunit were methylated: L7, L9, L11, L12, L18, and L33. Methylation of several other 50S proteins (such as L1, L3, L5, etc.) may also occur. The methylated amino acids in protein L11 have been characterized further and found to be predominately epsilon-trimethyllysine. A small amount of a compound tentatively identified as N(G), N'(G)-dimethylarginine was also detected.     

Comparative proteome analysis of thalamus in MK-801-treated rats

Two-dimensional gel-electrophoresis in combination with mass spectrometry is a powerful approach to compare protein expression in brain tissues. Using this proteomic approach, and based on the hypothesis that schizophrenia involves hypoglutamergic brain function, alterations in protein levels in the thalamus of rats treated with the N-methyl-D-aspartate (NMDA) receptor antagonist [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801), as compared to saline-treated animals, were assessed in an unbiased fashion. The rats were divided into two groups; group 1 (short-term treated) and group 2 (long-term treated). In group 1, the levels of seven proteins were increased and four proteins reduced. In group 2, the levels of six proteins were reduced. Several of the altered proteins (heat shock proteins 60 and 72, albumin, dihydropyrimidinase related protein-2, aldolase c, and malate dehydrogenase) have previously been connected to schizophrenia. Alterations of other proteins (dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2, guanine deaminase, alpha-enolase, aconitase, ATP-synthase and alpha-internexin), have not, to the best of our knowledge, earlier been implicated in schizophrenia pathology. Our results show the high potential of using proteomic methods for the validation of animal models of schizophrenia and to identify new proteins involved in the pathophysiological mechanisms of schizophrenia.     

Fabs enable single particle cryoEM studies of small proteins

In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ～65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.     

The Genetic Algorithm Applied as a Modelling Tool to Predict the Fold of Small Proteins with Different Topologies

The genetic algorithm exploits the principles of natural evolution. Solution trials are evolved by mutation, recombination and selection until they achieve near optimal solutions [1].Our own approach has now been developed [2] after a general overview on the application potential for protein structure analysis [3] to a tool to delineate the three-dimensional topology for the mainchain of small proteins [4], no matter whether they are largely helical, are mixed or -strand rich [5].Results on several protein examples for these different modelling tasks are presented and compared with the experimentally observed structures (RMSDs are around 4.5-5.5 Å). To start a modelling trial only the protein sequence and knowledge of its secondary structure is required. The fittest folds obtained after the evolution at the end of the simulations yield the three dimensional models of the fold. Current limitations are protein size (generally less than 100 aminoacids), number of secondary structure elements [7-8] and irregular topologies (e.g. ferridoxins).Further, preliminary results from current simulations are illustrated. We now want to apply simple experimental or other information, which is available long before the three-dimensional structure of the protein becomes known, to refine the modelling of the protein fold and tackle also more difficult modelling examples by our tool.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020304     

Fluorescence photobleaching recovery measurement of protein absolute diffusion constants

The technique of fluorescence photobleaching recovery [Axelrod et al., Biophys. J. 16 (1976) 1055] has been applied to the measurement of absolute diffusion constants of a number of fluoiescein isothiocyanate-labeled proteins. Measured diffusion constants agree to within +/- 7% of published values for the underivatized proteins. The method has sufficient sensitivity to reveal the concentration dependence at neutral pH of the diffusion constant of alpha-chymotrypsin. The rapidity with which the labelling and measurements can be performed and the small amount of material required suggest the technique may be useful in rapid characterization of small protein samples. Some developments in optical and electronic systems and in data processing for this technique are discussed.     

Engineered lanthanide-binding metallohomeodomains: designing folded chimeras by modular turn substitution

A series of chimeric metallohomeodomains are described, engineered by rational design of a flexible Ca/Ln binding site into a DNA-binding scaffold. A modular turn-substitution approach was used to create proteins that both bind DNA and lanthanide ions, while retaining the secondary structure of the full homeodomain (determined by circular dichroism [CD]). Four similar metallohomeodomains were designed (C1-C4), their structural stability predicted by molecular dynamics (MD) simulation of loop-mutations into the known homeodomain structure, and each designed protein cloned, expressed, and purified using standard molecular biology techniques. Two of the four loop insertions resulted in folded, metal- and DNA-binding proteins (EuC2 Kd = 2.1 +/- 0.4 microM; EuC4 Kd = 3.2 +/- 1.0 microM). These results show the successful incorporation of a metal site into a full protein domain, without compromising long-range structure. This is an important achievement in biomolecular design, as it provides a critical starting point for exploring metallonuclease function and substrate accessibility in a well-organized chimeric protein domain (rather than only in small HTH peptide systems).