Protein & Peptide Letters

ISSN: 0929-8665

Protein & Peptide Letters
Volume 13, Number 4, 2006

Contents


Regular Papers

Unfolding During Urea Denaturation of a Low Molecular Weight Phytocystatin (Thiol Protease Inhibitor) Purified from Phaseolus mungo (Urd) Pp. 323-329
S. Sharma, F. Rashid and B. Bano
[Abstract]


Cyclization of α-Synuclein Derived Peptide Increases Its Chaperone-Like Activity Pp. 331-333
T.D. Kim
[Abstract]


NMR Characterization of Recombinant Transmembrane Protein CB2 Fragment CB2_180-233 Pp. 335-342
J. Zhao, H. Zheng and X.-Q. Xie
[Abstract]


Production and Purification of Recombinant Human Glucagon Overexpressed as Intein Fusion Protein in Escherichia coli Pp. 343-347
R.S. Esipov, V.N. Stepanenko, A.I. Gurevich, L.A. Chupova and A.I. Miroshnikov
[Abstract]


The Odorant-Binding Protein from Canis familiaris: Purification, Characterization and New Perspectives in Biohazard Assessment Pp. 349-352
S. D’Auria, M. Staiano, A. Varriale, V. Scognamiglio, M. Rossi, A. Parracino, S. Campopiano, N. Cennamo and L. Zeni
[Abstract]


Expression of Active α-N-Acetylgluco-saminidase/TAT Chimerae in Cultured Spodoptera frugiperda Cells Pp. 353-356
J.C. Bandsmer, T.N. Campbell, I. Cheyne and F.Y.M. Choy
[Abstract]


Tyrosine Sulfation of Arylsulfatase A and Its Peptide Pp. 357-361
C. Kasinathan, S. Jean and P. Manowitz
[Abstract]


Purification and Properties of a Bovine Uricase Pp. 363-368
M.I. Rajoka, Khalil-ur-Rehman, M. Mehraj, M.W. Akhtar and M.A. Zia
[Abstract]


Expression, Purification and Characterization of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK I) Domain Expressed in Sf-9 Insect Cells Pp. 369-376
S.S. Khandekar, T. Yi, E. Dul, L.L. Wright, S. Chen, G.F. Scott, G.K. Smith, D. Lee, E. Hu and R.B. Kirkpatrick
[Abstract]


Fate of Influenza A Virus Proteins Pp. 377-384
G. Wu and S. Yan
[Abstract]


The Early Events of α-Synuclein Oligomerization Revealed by Photo-Induced Cross-Linking Pp. 385-390
H.-T. Li, X.-J. Lin, Y.-Y. Xie and H.-Y. Hu
[Abstract]


Network Analysis of the Protein Chain Tertiary Structures of Heterocomplexes⁺ Pp. 391-396
J.-J. Li, D.-S. Huang, T.-M. Lok, M.R. Lyu, Y.-X. Li and Y.-P. Zhu
[Abstract]


Leukocyte Function-Associated Antigen-1: Structure, Function and Application Prospects Pp. 397-400
M. Xingyuan, Z. Wenyun and W. Tianwen
[Abstract]


Comparative Study of Apoptosis Induced by H₂O₂ and NO: Limitation of Apoptosis Induced by NO Due to Slower Recovery of Activity of NO-Modified Caspase Pp. 401-404
J.E. Kim, H. Seok, P. Karki, J.S. Lee, S.Y. Shin, B. Cho and I.-S. Park
[Abstract]


Purification and Characterization of Arginine Kinase from Locust Pp. 405-410
M. Li, X.-Y. Wang and J.-G. Bai
[Abstract]


Purification and Properties of Laticeptin, an Antimicrobial Peptide from Skin Secretions of the South American Frog Leptodactylus laticeps Pp. 411-415
J.M. Conlon, N. Al-Ghaferi, B. Abraham, A. Sonnevend, J.D. King and P.F. Nielsen
[Abstract]


Crystallization Report

Crystallization and Preliminary X-Ray Crystallographic Analysis of Yeast Tyrosyl-tRNA Synthetase Complexed with Its Cognate tRNA Pp. 417-419
Y. Kusakabe, S. Ohno, N. Tanaka, M. Nakamura, M. Tsunoda, T. Moriguchi, N. Asai, M. Sekine, T. Yokogawa, K. Nishikawa and K.T. Nakamura
[Abstract]




Abstracts

[Back to top]
Unfolding During Urea Denaturation of a Low Molecular Weight Phytocystatin (Thiol Protease Inhibitor) Purified from Phaseolus mungo (Urd)
S. Sharma, F. Rashid and B. Bano

In the present study, two phytocystatins were purified to homogeneity as peaks I and II with molecular weights of 19 kDa and 17 kDa, respectively, as determined by SDS-PAGE and mass spectrometry. Both PMCs I and II were purified with a greater than 1000-fold purification and overall yield of about 16-18%. The effect of urea on PMC I and II was analysed by fluorescence and Circular Dichroism (CD) spectroscopy. Fluorescence studies suggest a red shift of the maximum emission at higher urea concentrations. PMC I and II are extremely stable protein inhibitors with regards to temperature and pH stability. FTIR studies show predominant α-helical structure in both the cystatins. CD analysis results show change in urea concentration-dependent loss in ellipticity, as well as in the shape of the CD spectrum compared to the intact phytocystatin.


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Cyclization of α-Synuclein Derived Peptide Increases Its Chaperone-Like Activity
T.D. Kim

We have analyzed a series of peptides derived from the C-terminus of α-synuclein for chaperone-like activity. Specifically, a cyclic peptide generated by introducing a disulfide bond was observed to increase chaperone-like activity. This is the first example of a disulfide-crosslinked peptide that exhibits activity against protein aggregation and activity loss.


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NMR Characterization of Recombinant Transmembrane Protein CB2 Fragment CB2_180-233
J. Zhao, H. Zheng and X.-Q. Xie

The expression of membrane proteins has been the bottleneck for their structural studies. Recently, we developed a method to obtain milligram quantities of isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor fragment in E. coli. In order to verify this method and confirm the recombinant isotope-labeled CB2 fragment, 3D hetero-nuclear NMR techniques were used to analyze the structure of the fragment CB2_180-233 in DMSO-d6 solvent. The sequential assignments of TM5 and intra-cellular loop 3 were accomplished, which confirmed the experimental protocols of isotope-labeled recombinant protein expression, fusion protein cleavage, and membrane protein purification. The obtained structure also showed α-helix in the TM5 region, but it was interrupted by a disordered region (Gly204_ILe206). These results further revealed that our established approach is a promising method to express recombinant membrane proteins for their structural studies.


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Production and Purification of Recombinant Human Glucagon Overexpressed as Intein Fusion Protein in Escherichia coli
R.S. Esipov, V.N. Stepanenko, A.I. Gurevich, L.A. Chupova and A.I. Miroshnikov

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.


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The Odorant-Binding Protein from Canis familiaris: Purification, Characterization and New Perspectives in Biohazard Assessment
S. D’Auria, M. Staiano, A. Varriale, V. Scognamiglio, M. Rossi, A. Parracino, S. Campopiano, N. Cennamo and L. Zeni

In this report we show the purification to homogeneity and a partial characterization of a new odorant-binding protein from Canis familiar (CfOBP) nasal mucosa. In addition, we report preliminary data on the utilization of CfOBP as a probe for the development of a refractive index-based biosensor.


[Back to top]
Expression of Active α-N-Acetylgluco-saminidase/TAT Chimerae in Cultured Spodoptera frugiperda Cells
J.C. Bandsmer, T.N. Campbell, I. Cheyne and F.Y.M. Choy

We examined the production and secretion of fusion constructs containing α-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/α-N-acetylglucosaminidase chimerae in enzyme replacement therapy.


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Tyrosine Sulfation of Arylsulfatase A and Its Peptide
C. Kasinathan, S. Jean and P. Manowitz

Purified human liver arylsulfatase A (ASA) as well as an ASA peptide (residues 28-39) were sulfated by tyrosyl protein sulfotransferase in vitro. The media, but not the cell lysate, of normal human fibroblasts contained a tyrosine sulfated protein (pI = 4.5-5.5). This protein was not present in either media or cell lysate of human fibroblasts lacking ASA protein. These results suggest that tyrosine sulfation facilitates secretion of ASA and that this may have pathophysiological consequences.


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Purification and Properties of a Bovine Uricase
M.I. Rajoka, Khalil-ur-Rehman, M. Mehraj, M.W. Akhtar and M.A. Zia

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K_m and V_max values for uric acid hydrolysis were 0.125 mM and 102 IU mg^-1 protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy Δ H*) and entropy of activation (Δ S*) for uricase activity were lower than those reported for some thermostable enzymes.


[Back to top]
Expression, Purification and Characterization of an Enzymatically Active Truncated Human Rho-Kinase I (ROCK I) Domain Expressed in Sf-9 Insect Cells
S.S. Khandekar, T. Yi, E. Dul, L.L. Wright, S. Chen, G.F. Scott, G.K. Smith, D. Lee, E. Hu and R.B. Kirkpatrick

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.


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Fate of Influenza A Virus Proteins
G. Wu and S. Yan

In this review we summarize the current state, history, future, mutation tendency and species susceptibility of influenza A virus proteins based on our probabilistic analyses on amino acid pairs, and compare the current state of influenza A virus proteins with that of proteins which we have studied in the past.


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The Early Events of α-Synuclein Oligomerization Revealed by Photo-Induced Cross-Linking
H.-T. Li, X.-J. Lin, Y.-Y. Xie and H.-Y. Hu

Assembly of α-synuclein (α-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson’s disease. Studying the early events of α-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of α-Syn. Results show that α-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of α-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oli-gomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of α-Syn aggregation and the neurotoxic aggregation species.


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Network Analysis of the Protein Chain Tertiary Structures of Heterocomplexes_⁺
J.-J. Li, D.-S. Huang, T.-M. Lok, M.R. Lyu, Y.-X. Li and Y.-P. Zhu

In this paper, the tertiary structures of protein chains of heterocomplexes were mapped to 2D networks; based on the mapping approach, statistical properties of these networks were systematically studied. Firstly, our experimental results confirmed that the networks derived from protein structures possess small-world properties. Secondly, an interesting relationship between network average degree and the network size was discovered, which was quantified as an empirical function enabling us to estimate the number of residue contacts of the protein chains accurately. Thirdly, by analyzing the average clustering coefficient for nodes having the same degree in the network, it was found that the architectures of the networks and protein structures analyzed are hierarchically organized. Finally, network motifs were detected in the networks which are believed to determine the family or superfamily the networks belong to. The study of protein structures with the new perspective might shed some light on understanding the underlying laws of evolution, function and structures of proteins, and therefore would be complementary to other currently existing methods.


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Leukocyte Function-Associated Antigen-1: Structure, Function and Application Prospects
M. Xingyuan, Z. Wenyun and W. Tianwen

This review focuses on the structure, function and pathological role of leukocyte function-associated antigen-1 (LFA-1) which is a heterodimeric protein consisting of two subunits. LFA-1 plays a most important role in the immune system including adhesion, extravasation, migration, apoptosis, cytotoxicity, cytokine production, and proliferation of lymphocytes. Therefore, T-cell activation can be suppressed by blocking ICAM-1/LFA-1 interaction in autoimmune diseases and organ transplantation. Many different inhibitors (i.e. antibodies, peptides, small molecules) have been demonstrated to block ICAM-1/LFA-1 interaction, and some of them are promising for medical treatment or have reached clinical trials.


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Comparative Study of Apoptosis Induced by H₂O₂ and NO: Limitation of Apoptosis Induced by NO Due to Slower Recovery of Activity of NO-Modified Caspase
J.E. Kim, H. Seok, P. Karki, J.S. Lee, S.Y. Shin, B. Cho and I.-S. Park

Both H₂O₂ and NO can act as apoptogens, triggering apoptosis in many cells. They are also well known inhibitors of caspases, essential enzymes in apoptosis. The differences between these two agents as apoptosis inducers and how caspases mediate apoptosis with these inhibitory agents is still unclear. Consistent with the previous reports, these two agents induced apoptosis accompanied by caspase activation with limitation of all apoptotic events for NO. It was found that NO-modified caspase-3 showed a slower recovery of its activity in the presence of the reducing agents compared to that of H₂O₂ modification. This is one possible cause of the limited apoptosis in the case of NO.


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Purification and Characterization of Arginine Kinase from Locust
M. Li, X.-Y. Wang and J.-G. Bai

L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30°C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k_cat/K_m^Arg 174. The activity of AK required divalent cations such as Mg²⁺ and Mn²⁺. In the presence of Cu²⁺ and Zn²⁺, AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.


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Purification and Properties of Laticeptin, an Antimicrobial Peptide from Skin Secretions of the South American Frog Leptodactylus laticeps
J.M. Conlon, N. Al-Ghaferi, B. Abraham, A. Sonnevend, J.D. King and P.F. Nielsen

Norepinephrine-stimulated skin secretions from the Sante Fe frog Leptodactylus laticeps contained high concentrations of a peptide, termed laticeptin, with the primary structure Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Leu-Ala-Gly-His-Leu-Ala-Thr-Lys-Val-Met-Asn-Lys-Leu.NH₂. Laticeptin inhibited the growth of selected Gram-negative bacteria but the lack of activity against Gram-positive bacteria and the very low hemolytic activity is probably a consequence of the weak amphipathicity of the peptide in its α-helical conformation.


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Crystallization and Preliminary X-Ray Crystallographic Analysis of Yeast Tyrosyl-tRNA Synthetase Complexed with Its Cognate tRNA
Y. Kusakabe, S. Ohno, N. Tanaka, M. Nakamura, M. Tsunoda, T. Moriguchi, N. Asai, M. Sekine, T. Yokogawa, K. Nishikawa and K.T. Nakamura

Yeast tyrosyl-tRNA synthetase (yTyrRS) has been crystallized by the vapor diffusion method in the presence of its cognate tRNA^Tyr. The crystals belong to a tetragonal space group P4₁2₁2 with cell dimensions of a = b = 63.85 Å, and c = 330.3 Å. The asymmetric unit contains one molecule each of yTyrRS and tRNA^Tyr (one-half of a 2:2 complex). X-ray diffraction data have been collected up to 2.5 Å resolution.