Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

Evaluation of methods for the separation and analysis of proteins and free amino acids in phytoplankton samples

Due to their importance as not only major constituents in paniculatematter but also the metabolism of nitrogen in marine microorganisms,numerous methods have been employed to measure proteins andfree amino acids. However, two difficulties frequently complicatethese measurements. First, an initial separation of proteinsfrom free amino acids is helpful since most analytical methodsare somewhat sensitive to both compound types. Second, the choiceof detection techniques that minimize response differences betweenvarious proteins or amino acids is desirable since natural samplesof microorganisms consist of mixtures of many proteins and aminoacids. To address these problems, four protein detection techniques(modified Lowry et al., Dorsey et al., Bradford and fluorescamine)and two amino acid detection techniques (fluorescamine and o-phthaldialdehyde)were evaluated. Relative extraction efficiencies for proteinfrom phytoplankton samples were also evaluated with six homogenizationsolutions/protocols (TCA, NaOH, boiling NaOH, Triton X-100,NaOH plus Triton X-100 and distilled water). TCA homogenizationyielded the highest protein recoveries, and sufficient physicalseparations between proteins and free amino acids were obtainedwith TCA concentrations between 0.18 and 0.37 M. Results ofthese studies allowed for development of a method for extracting,separating and analyzing proteins and total free amino acidsfrom a common phytoplankton sample. The procedure involves initialhomogenization in a TCA solution, followed by centrifugationto separate protein and free amino acid fractions. Proteinsare then analyzed by a modification of the Lowry et al. procedure,and amino acids by a fluorescamine procedure. ²Present address: Science Applications International Corporation,4224Campus Point Court, San Diego, CA 92121, USA     

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.     

The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.     

Efficient identification of amino acid types for fast protein backbone assignments

We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective ¹⁵N-labeling. The usefulness of selective incorporation of ¹⁵N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with ¹⁵N-labeled amino acids. Here we demonstrate that purification of the selectively ¹⁵N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins.     

A simple, sensitive radiomicroassay for 5'-nucleotidase

The reagent 9,10-phenanthrenequinone has been shown to react with free arginine or with arginine residues within proteins to produce a compound whose fluorescence can be used to quantitatively determine submicrogram amounts of arginine. The assay procedure, which is simple, convenient, and suitable for automation, is performed by mixing a slight excess of phenanthrenequinone with the sample at high pH followed by acidification to produce the fluorescence. None of the commonly occurring amino acids were found to interfere with the analysis. Several commonly used buffers and organic solvents also did not interfere. The arginine content of intact proteins was accurately determined by this procedure with only microgram quantities of protein required. The method is compared with other commonly used procedures for arginine and protein determination.     

Circular dichroism studies on synthetic peptides corresponding to the cleavage site region of precursor proteins

The conformations of synthetic peptides which span the region in which the precursor part of proteins (signal sequences) destined for export are cleaved by signal peptidases, were investigated by circular dichroism spectroscopy. Pentapeptides comprising amino acids only from the carboxy-terminus of signal sequences or the amino terminus of the mature protein do not have any preferred conformation in a variety of solvents. Octa- and nonapeptides containing amino acids from the carboxy-terminal protion of signal sequences and the amino-terminus of the mature portions of precursor proteins tend to adopt beta-turn conformations in trifluoroethanol and micelles of sodium dodecylsulphate. Hence, in addition to the distribution of amino acids with small side chains at the carboxy terminus of signal sequences, it is conceivable that signal peptidases also recognize a beta-turn conformation in the cleavage site region of precursor proteins.     

Application of the flame photometric sulfur detector to the gas-chromatographic analysis of trimethylsilylated methylthiohydantoins of amino acids

Application of the flame photometric detector was made to the gasliquid chromatographic analysis of trimethylsilylated methylthiohydantoins. Principal advantage of the detector proved to be its specificity. The detector was sensitive to the trimethylsilyl derivatives of each of nineteen methylthiohydantoins studied, and extraneous peaks which were occasionally observed with the flame ionization detector were completely absent. Linear calibration curves (log peak area vs log amount chromatographed) were obtained for twelve of the methylthiohydantoins studied, and a rough quantitative estimate of the amount present could be made for the remaining seven.     

The determination of similarities in amino acid composition among proteins separated by two-dimensional gel electrophoresis

A simple and rapid procedure has been developed to determine similarities in amino acid composition among cellular proteins separated by two-dimensional gel electrophoresis. Cells in tissue culture are simultaneously labeled with two different amino acids each tagged with a different radioisotope. The proteins are then separated on two-dimensional gels and their location on the gels determined by Coomassie-blue staining or autoradiography. Elution of the protein from the appropriate region of the gel followed by liquid scintillation counting yields an isotope ratio which reflects the ratio of the two amino acids in the protein. Examples of the use of this technique in analyzing mutant proteins, proteins altered by carbamylation, and cell proteins with similar amino acid composition (e.g., actin and tubulin) are given.     

Presence of a characteristic D-D-E motif in IS1 transposase

Transposases encoded by various transposable DNA elements and retroviral integrases belong to a family of proteins with three conserved acidic amino acids, D, D, and E, constituting the D-D-E motif that represents the active center of the proteins. IS1, one of the smallest transposable elements in bacteria, encodes a transposase which has been thought not to belong to the family of proteins with the D-D-E motif. In this study, we found several IS1 family elements that were widely distributed not only in eubacteria but also in archaebacteria. The alignment of the transposase amino acid sequences from these IS1 family elements showed that out of 14 acidic amino acids present in IS1 transposase, three (D, D, and E) were conserved in corresponding positions in the transposases encoded by all the elements. Comparison of the IS1 transposase with other proteins with the D-D-E motif revealed that the polypeptide segments surrounding each of the three acidic amino acids were similar. Furthermore, the deduced secondary structures of the transposases encoded by IS1 family elements were similar to one another and to those of proteins with the D-D-E motif. These results strongly suggest that IS1 transposase has the D-D-E motif and thus belongs to the family of proteins with the D-D-E motif. In fact, mutant IS1 transposases with an amino acid substitution for each of the three acidic amino acids possibly constituting the D-D-E motif were not able to promote transposition of IS1, supporting this hypothesis. The D-D-E motif identified in IS1 transposase differs from those in the other proteins in that the polypeptide segment between the second D and third E in IS1 transposase is the shortest, 24 amino acids in length. Because of this difference, the presence of the D-D-E motif in IS1 transposase has not been discovered for some time.     

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320- 322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.     

Comparison of the frequency of functional SH3 domains with different limited sets of amino acids using mRNA display

Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory.     

N- and C-terminal amino acids of proteolipids from the rat brain and various other organs

N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.     

低温对螺旋藻可溶性蛋白和游离氨基酸外渗的影响

以鄂尔多斯高原碱湖特有的钝顶螺旋藻S1、引进的钝顶螺旋藻S2和极大螺旋藻S3为材料,采用Folin法和茚三酮法分别测定低温下细胞外渗液中可溶性蛋白和游离氮基酸含量。结果表明：处理温度越低,时间越长,螺旋藻细胞外渗液中可溶性蛋白和游离氨基酸的含量越高。S1膜损伤程度最轻,比引进种S2、S3有较强的抗寒能力。判断低温对螺旋藻膜损伤的程度,用可溶性蛋白较游离氨基酸更接近于实际。     

The exchangeability of amino acids in proteins

The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of "experimental exchangeability" values EX(ij) derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance.     

Amino acid sequence of the gene 0.3 protein of bacteriophage T7 and nucleotide sequence of its mRNA

The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.     

Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G) contains 20 uncharged and mostly hydrophobic amino acids. We created DNAs specifying G proteins with shortened transmembrane domains, by oligonucleotide-directed mutagenesis. Expression of these DNAs showed that G proteins containing 18, 16, or 14 amino acids of the original transmembrane domain assumed a transmembrane configuration and were transported to the cell surface. G proteins containing only 12 or 8 amino acids of this domain also spanned intracellular membranes, but their transport was blocked within a Golgi-like region in the cell. A G protein completely lacking the membrane-spanning domain accumulated in the endoplasmic reticulum and was secreted slowly. These experiments indicate that the size of the transmembrane domain is critical not only for membrane anchoring, but also for normal cell surface transport.     

Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system.

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.     

The sharing of amino acid short spans by ancestrally unrelated proteins may be the result of ubiquitous alpha and beta secondary structures

Short homologies are often found when genetically unrelated proteins are compared but it is not known whether the rate at which they occur is or not above randomness. Comparing 190 pairs of unrelated proteins enable us to show that the frequency at which pairs of unrelated proteins share little spans of amino acids is compatible with chance.However, it appears that those short homologies are mainly located within protein subregions of identical secondary structure: the frequency at which pairs of unrelated proteins exhibit related spans of amino acids inside subregions of identical secondary structure is far above randomness. Those data suggest that the sharing of related spans of amino acids by genetically unrelated proteins could result from structural constraints imposed by the α or β secondary structures.     

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.