Protein identification using 20-minute Edman cycles and sequence mixture analysis

We have developed a 20-min Edman cycle and a multiple sample horizontal flow reactor for the sequence analysis of PVDF-electroblotted proteins. The 20-min cycle uses a 12-min C18 phenylthiohydantoin separation. This cycle and separation is compatible with most Applied Biosystems sequencers. Using this rapid cycle, 10 residues on six different proteins can be completed within a 24-h period. We also demonstrate the use of an algorithm that can sort mixture sequences derived from PVDF bands that contain coeluting proteins.     

Inverse sequence similarity in proteins and its relation to the three-dimensional fold

PURPOSE: To determine whether an expression vector that encodes for human tyrosinase, the key enzyme in the melanin production pathway, can be used to image gene expression with magnetic resonance (MR) imaging and scintigraphy. MATERIALS AND METHODS: Mouse fibroblasts and human embryonal kidney cells were transfected with an expression vector that contained a complete complementary DNA sequence that encodes the human tyrosinase gene (pcDNA3tyr). Transfected cells were assayed for messenger RNA presence, melanin staining, and indium-111 binding; scintigraphy and MR imaging were performed. RESULTS: Transfected cells contained tyrosinase messenger RNA and stained positively for melanin. Transfected cells had a higher In-111 binding capacity than nontransfected cells, a difference readily detectable with scintigraphy. MR imaging showed transfected cells to have markedly higher signal intensity after gene transfer than nontransfected cells. CONCLUSION: Gene transfer and expression in cell culture can be detected with MR imaging and scintigraphy. The proposed strategy of using an imaging marker gene may have a substantial effect on the noninvasive imaging of gene therapy.     

Protein sequence similarity searches using patterns as seeds

Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.     

Protein Motifs. 7. The four-helix bundle: what determines a fold?

The four-helix bundle motif occurs in many structural contexts and in proteins that are functionally diverse. The motif can be classified into individual folds on the basis of topological and geometric properties. It has been thoroughly investigated structurally by both nuclear magnetic resonance and x-ray crystallography. Many mutants of four-helix bundles have been generated, and the motif has also been the target of de novo design studies. Taken together, these studies provide an opportunity to examine many of the forces governing protein folding. In this article we consider the relative importance of the burial of hydrophobic residues, loss of conformational entropy, packing interactions, interhelical turn composition, and helical dipole interactions all within the context of a single folding motif. We conclude by examining why de novo designed four-helix bundle proteins possess flexible interiors, and possible mechanisms by which natural proteins may lock their cores into rigid structures.     

Pitfalls of protein sequence analysis

Thirteen patients who developed restrictive subdeltoid adhesions after rotator cuff repair were identified. These patients underwent second-look arthroscopy and takedown of adhesions at an average of 37 weeks after their index surgery. Clinical findings include pain and restricted motion that does not yield to manipulation under anesthesia. Arthroscopic findings are subdeltoid adhesions and a chondral lesion (companion lesion) of the humeral head articular surface. Patients were reevaluated at 26 weeks after their release of adhesions. Prerelease and postrelease University of California, Los Angeles (UCLA) scores average 14.8 and 30.1, respectively. Prerelease and postrelease UCLA pain scores averaged 2.6 and 7.7, respectively. Prerelease and postrelease range of motion was as follows: Flexion, 141/158; abduction, 123/141; internal rotation, 47/69; and external rotation, 53/74. The authors have proposed a theory to explain the clinical and arthroscopic findings in this subgroup of patients who are dissatisfied after rotator cuff repair. A technique for and the results of release of the subdeltoid adhesions also are reported.     

Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation

The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.     

热轧薄规格钢带折叠缺陷成因分析及措施探讨

针对热轧折叠缺陷对酸连轧工序造成的原料空卷问题,从热轧工序相关影响因素出发,分析了薄规格热轧钢带生产过程中折叠缺陷形成原因,主要为钢带头尾存在不同程度镰刀弯或局部浪形等问题,经卷取侧导板和夹送辊共同挤压作用后形成的。通过在设备精度、工艺改进、预警机制等方面进行系统优化和改进,薄规格热轧钢带折叠缺陷发生量得以有效控制,月均发生量从攻关前的55卷左右逐渐下降至攻关以来的20卷左右水平。进一步提高了热轧钢带一检合格率和折叠缺陷的预警识别率,有效降低了热轧钢带折叠类缺陷对下游工序生产过程造成的影响,促进了上下游生产节奏的提升。     

Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Artificial neural networks for molecular sequence analysis

Artificial neural networks provide a unique computing architecture whose potential has attracted interest from researchers across different disciplines. As a technique for computational analysis, neural network technology is very well suited for the analysis of molecular sequence data. It has been applied successfully to a variety of problems, ranging from gene identification, to protein structure prediction and sequence classification. This article provides an overview of major neural network paradigms, discusses design issues, and reviews current applications in DNA/RNA and protein sequence analysis.     

DNA sequence analysis by MALDI mass spectrometry

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.     

A nonlinear operator-based speech feature analysis method with application to vocal fold pathology assessment

Traditional speech processing methods for laryngeal pathology assessment assume linear speech production with measures derived from an estimated glottal flow waveform. They normally require the speaker to achieve complete glottal closure, which for many vocal fold pathologies cannot be accomplished. To address this issue, a nonlinear signal processing approach is proposed which does not require direct glottal flow waveform estimation. This technique is motivated by earlier studies of airflow characterization for human speech production. The proposed nonlinear approach employs a differential Teager energy operator and the energy separation algorithm to obtain formant AM and FM modulations from filtered speech recordings. A new speech measure is proposed based on parameterization of the autocorrelation envelope of the AM response. This approach is shown to achieve impressive detection performance for a set of muscular tension dysphonias. Unlike flow characterization using numerical solutions of Navier-Stokes equations, this method is extremely computationally attractive, requiring only a small time window of speech samples. The new noninvasive method shows that a fast, effective digital speech processing technique can be developed for vocal fold pathology assessment without the need for direct glottal flow estimation or complete glottal closure by the speaker. The proposed method also confirms that alternative nonlinear methods can begin to address the limitations of previous linear approaches for speech pathology assessment.     

Systematic fold recognition analysis of the sequences encoded by the genome of Mycoplasma pneumoniae

A robust tool for fold recognition was applied to the systematic analysis of the sequences below 200 residues encoded by the genome of Mycoplasma pneumoniae. The goal was to determine the additional information gain achievable in genome analysis by fold recognition, beyond the intrinsic limits of homology studies. A list of 124 sequences encoding for soluble proteins or domains not homologous to each other, or to proteins with known three-dimensional structure, was analyzed, resulting in significant Z scores for the energy of the structural models in 12 of these cases. This result indicates that systematic application of fold recognition techniques to the analysis of structurally unassigned soluble proteins can lead to high-confidence structural predictions with an efficiency of about 10%, a relevant contribution besides the complementary approach of homology analysis. Four of the predictions presented include mapping of the putative active site of the target sequence and lead to the detection of probable catalytic and binding residues. The data are discussed with reference to the functional implications of the structural models and to the results reported for the homologous genome of Mycoplasma genitalium.     

Protein structure and energy landscape dependence on sequence using a continuous energy function

We have recently described a new conformational search strategy for protein folding algorithms called the CGU (convex global underestimator) method. Here we use a simplified protein chain representation and a differentiable form of the Sun/Thomas/Dill energy function to test the CGU method. Standard search methods, such as Monte Carlo and molecular dynamics are slowed by kinetic traps. That is, the computer time depends more strongly on the shape of the energy landscape (dictated by the amino acid sequence) than on the number of degrees of freedom (dictated by the chain length). The CGU method is not subject to this limitation, since it explores the underside of the energy landscape, not the top. We find that the CGU computer time is largely independent of the monomer sequence for different chain folds and scales as O(n4) with chain length. By using different starting points, we show that the method appears to find global minima. Since we can currently find stable states of 36-residue chains in 2.4 hours, the method may be practical for small proteins.