[14] Measurements of protein hydration by various techniques

Pessen, Helmut; Kumosinski, Thomas F.

doi:10.1016/s0076-6879(85)17016-3

Cited by 67 publications

(61 citation statements)

References 52 publications

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“…The coefficient was corrected to standard conditions to obtain the corresponding standardized sedimentation coefficient (S 20,w ) value using the SEDNTERP program (33). The translational frictional coefficient of MobMN243 (f) was determined from the molecular mass and sedimentation coefficient of the protein (34), whereas the frictional coefficient of the equivalent hydrated sphere (f 0 ) was estimated using a hydration of 0.4506 g H 2 O per g of protein (35).…”

Section: Methodsmentioning

confidence: 99%

Functional Properties and Structural Requirements of the Plasmid pMV158-Encoded MobM Relaxase Domain

et al. 2013

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A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriT pMV158 , whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N-and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself. C onjugation is one of three fundamentally different ways, in addition to transformation and transduction, by which the genetic content of a cell can be altered (1, 2). The conjugative process involves active DNA transfer from one living organism to another through the type 4 secretion system (T4SS), which is encoded on the donor cell side (3, 4). The shuttling of plasmids and integrative and conjugative elements (ICEs) between organisms has proven to be a major contributor to evolutionary diversity, as many genes have been exchanged rather than evolved by mutation during evolution (5). Although conjugation is generally associated with the exchange of genetic material, it most probably originated as a protein transport mechanism, since DNA transfer is always preceded by protein transfer and T4SS transfer is frequently used for translocation of proteinaceous virulence factors (6). The protein that plays a central role in the conjugational transfer of DNA is termed relaxase (7,8). It associates with other factors to form a complex called the relaxosome (8), which provides the context for the controlled cleavage and processing of transferable DNA. The relaxase recognizes a specific DNA sequence, the origin of transfer (oriT), which generally contains one or more inverted repeats (9). The relaxase then nicks the DNA at a specific position and forms a stable complex with the oriT-harboring strand in the 5= terminus, leaving a free 3=-OH end. The DNA-pro...

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Section: Methodsmentioning

confidence: 99%

Functional Properties and Structural Requirements of the Plasmid pMV158-Encoded MobM Relaxase Domain

et al. 2013

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“…f values for bFGF and the bFGF⅐PF4-(47-70) complex were calculated from their molecular masses and from their s 20,w coefficients. f 0 was estimated using a hydration coefficient of 0.3 g of H 2 O/g of protein (58). The frictional coefficient ratio (f/f 0 ) allows the calculation of a family of ellipsoids of revolution compatible with the hydrodynamic properties of the protein (59,60).…”

Section: Methodsmentioning

confidence: 99%

Solution Structure and Interaction with Basic and Acidic Fibroblast Growth Factor of a 3-kDa Human Platelet Factor-4 Fragment with Antiangiogenic Activity

Lozano¹,

Redondo‐Horcajo²,

Jiménez³

et al. 2001

Journal of Biological Chemistry

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Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue Cterminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factordriven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.Angiogenesis plays an important role during embryonic development and in many pathologies including cancer and cardiovascular and inflammatory diseases. Fibroblast growth factors (FGFs) 1 and vascular endothelial growth factor (VEGF) family members constitute the major class of angiogenic regulators. These factors induce endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo (1, 2). FGFs and VEGFs induce specific cellular responses by binding selectively to cell-surface receptors. The FGF family is presently known to include 22 closely related proteins. Acidic fibroblast growth factor (aFGF) and the subsequently discovered basic fibroblast growth factor (bFGF) are considered to be representative of the whole family (3-5). FGFs bind to two receptor classes with high and low affinity at the cell surface. The high affinity receptors (FGFR) belong to the class of the transmembrane receptor tyrosine kinases (6). FGFRs are widely distributed in all cells of mesodermal and neuroectodermal origin studied thus far, which accounts for the wide spectrum of in vitro mitogenic targets of this protein family (7,8). The low affinity receptor of FGFs is the glycosidic moiety of the cell coat and basal membranes (9). Angiogenesis promoted by VEGF has been reported to be dependent on the endogenous expression of bFGF by endothelial cells and is therefore blocked by antibodies that neutralize to bFGF (10, 11). Consequently, there is considerable pharmacological interest in developing inhibitors with the mitogenic activity of FGFs. Platelet factor-4 (PF4) is a CXC chemokine with high affinity for sulfated glycosaminoglycans like heparin and heparan sulfate. PF4 inhibits endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo (12)(13)(14). Furthermore, th...

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“…The independently determined molecular weight (from sedimentation equilibrium) was used to estimate the frictional coefficient (f) from the Svedberg equation (S 20,w ϭ M(1 Ϫ ⅐)/N⅐f). The frictional coefficient of a sphere of equivalent mass (f o ), was calculated assuming a degree of hydration of 0.28 g of water/g of protein (26,27).…”

Section: Methodsmentioning

confidence: 99%

The N-terminal Region of Human Progesterone B-receptors

Bain

Franden

McManaman

et al. 2001

Journal of Biological Chemistry

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Progesterone receptors (PR) 1 are members of the nuclear receptor family of ligand-dependent transcription factors (1). In normal and malignant tissues there are two, naturally occurring, PR isoforms, 82-kDa A-receptors and 99-kDa B-receptors (2). The proteins are identical except for a 164-amino acid extension (BUS) at the N terminus of B-receptors. Each isoform contains two well characterized autonomous units: a C-terminal hormone binding domain (HBD), and a centrally located DNA binding domain (DBD). The two domains are linked by a short nuclear localization signal and a 50-amino acid "hinge" sequence of unclear function. Though critically important to activity, the DBD and HBD together comprise only 52 and 44% of the mass of the A-and B-receptors, respectively. The remaining sequences N-terminal to the DBD are poorly characterized. The N-terminal regions of both isoforms contain a 91-amino acid transcriptional activation domain (AF1) adjacent to the DBD (3). Additionally, B-receptors contain a context-dependent activation domain (AF3) within BUS (4). The 291-amino acid region between BUS and AF1 is transcriptionally inhibitory (5). Despite their close structural similarity, the two isoforms have pronounced functional differences on artificial promoters.

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[14] Measurements of protein hydration by various techniques

Cited by 67 publications

References 52 publications

Functional Properties and Structural Requirements of the Plasmid pMV158-Encoded MobM Relaxase Domain

Functional Properties and Structural Requirements of the Plasmid pMV158-Encoded MobM Relaxase Domain

Solution Structure and Interaction with Basic and Acidic Fibroblast Growth Factor of a 3-kDa Human Platelet Factor-4 Fragment with Antiangiogenic Activity

The N-terminal Region of Human Progesterone B-receptors

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