Characterization of monomeric and dimeric B domain of Staphylococcal protein A.

Both monomeric and dimeric constructs of the B domain of protein A from Staphylococcus aureus have been characterized by NMR, CD and fluorescence spectroscopy. The monomeric form of the protein was synthesized using a novel method incorporating the use of a recombinant, folded, chimeric protein. A comparison of the recombinant monomeric form with the commercially available dimeric form indicates that, although the dimer retains the integrity of the three-helix bundle structure present in the monomer, there are interdomain contacts in the dimeric form. A single long-lived water molecule in the hydrophobic core of the three-helix bundle of monomeric protein A may represent an important stabilizing factor for the three-helix bundle topology.     

Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features

The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C(HCV)169) and its N-terminal region from positions 1 to 117 (C(HCV)117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C(HCV)169 and C(HCV)117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C(HCV)169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of alpha-helices (50%). In contrast, C(HCV)117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.     

Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry.

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)3, a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS-PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal-ion binding to the protein species, which may alter the function and binding of the protein partners.     

Characterization of a dimeric canine form of surfactant protein C (SP-C)

Canine pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide which has one palmitoylated cysteine residue. SP-C enhances the insertion of phospholipids into a monolayer. Two forms of canine SP-C were isolated using Sephadex LH-60 chromatography. It was found that canine SP-C exists in a palmitoylated monomeric form of 3.5 kDa, and a non-acylated dimeric form of 7 kDa. Circular dichroism showed that both forms of SP-C exhibit similar secondary structures at the air/water interface. Both forms of SP-C were able to induce the insertion of phospholipids into a monolayer as measured with the Wilhelmy plate technique. In contrast to the palmitoylated monomeric form of SP-C, the non-acylated dimeric form of SP-C does not require calcium ions to insert phospholipids into a monolayer without the negatively charged phosphatidylglycerol. It is concluded that two forms of canine SP-C exist, but the physiological significance of these different forms remains to be established.     

Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A.

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.     

The helical model of the nucleosome core.

A model of the nucleosome core is proposed based on a topologically linear array of histones attached sequentially to DNA. The linear complex folds helically forming a spring-like particle. Different variants of the particle are discussed (cylindrical springs with and without histone-histone contacts between turns of the helix, solenoidal spring). The model is consistent with known data about the nucleosome structure. Histones H3 and H4 have a special role in the model which is related also to the superstructure of chromatin.     

Penicillin-binding protein 1B of Escherichia coli exists in dimeric forms.

A high-molecular-weight band has been detected in Western immunoblots of nonboiled Escherichia coli samples incubated with polyclonal antiserum against penicillin-binding protein 1B (PBP 1B). This band was shown to be a dimer of PBP 1B. The dimer was more strongly associated with the envelope than the monomer, and it was still able to bind penicillin G. Analysis of the binding of fusion proteins of PBP 1B and beta-lactamase showed that the part of PBP 1B necessary for complex formation lies in the amino-terminal half of the protein.     

Nebulin, a helical actin binding protein.

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a 'molecular ruler'. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F-actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin--actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F-actin-nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two-start helix. The consistency of this model with experimental data is discussed.     

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.     

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.     

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.     

Expression and characterization of minican, a recombinant syndecan-1 with extensively truncated core protein.

Syndecan-1 is an integral membrane heparan sulfate/chondroitin sulfate proteoglycan, involved in the control of cell growth and differentiation. The biological activities of syndecan-1 involve interactions with a variety of extracellular ligands, such as growth factors and matrix components, that are mainly mediated by the heparan sulfate moieties. The expression of syndecan-1 is downregulated in various malignant tumors, and low levels of expression appear to correlate with poor prognosis of some cancer types. On the other hand, the extracellular portion of syndecan-1 (ectodomain) has been demonstrated to inhibit the proliferation of various cancer cells in culture, suggesting that proteoglycan-like molecules should be studied further with regard to their antitumor activities. We have expressed, in CHO cells, a truncated syndecan-1 ectodomain ("minican") harboring domains for glycosaminoglycan attachment and antibody recognition. Analysis of recombinant minican indicates that it shares some of the biochemical and biological characteristics attributed to syndecan-1 ectodomain. Minican was thus substituted with heparan sulfate chains and bound to extracellular matrix proteins as well as fibroblast growth factors. Notably, minican inhibited the proliferation of S115 mouse mammary carcinoma cells and the effect seemed to involve inhibition of the Ras/Erk signaling pathway. Our data suggest that recombinant syndecan-1 with a minimal protein component is biologically active. This information may provide useful in further design of proteoglycan-like antitumor molecules.     

Immobilization of urease using Amberlite MB-1

Amberlite MB-1 was used to immobilize urease (EC.3.5.1.5). The thermal stability of the immobilized urease was better than that of the free urease. Its highest activity was obtained at 75?°C and at pH 6.5 while the optimum temperature for the free urease was found to be 25?°C. Urease immobilized on Amberlite MB-1 retained 65% of the original activity after 5 repeated uses and 62% of the activity after 60 days when stored at 4?°C.     

Staphylococcus aureus sortase A exists as a dimeric protein in vitro

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

Flavivirus capsid is a dimeric alpha-helical protein

The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. The oligomeric properties of the capsid protein in solution were investigated. In the absence of nucleic acid, both proteins were predominantly dimeric in solution. Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical. The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments. The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices. The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character. In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein.     

Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals

The incorporation of alpha-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib)3-OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven alpha-type hydrogen bonds in the middle and 3(10)-type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH...OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in alpha-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration. The space group is P2(1)2(1)2(1), with a = 9.964 (3) A, b = 20.117 (3) A, c = 39.311 (6) A, Z = 4, and dx = 1.127 g/cm3 for C64H106N13O16.1.33H2O. The final agreement factor R was 0.089 for 3667 data observed greater than 3 sigma(F) with a resolution of 0.9 A.