pH-dependent polymorphism of assemblies of cytokine hMIP-lβ

1994 ◽  
Vol 52 ◽  
pp. 434-435
Author(s):  
N. Cheng ◽  
A. M. Gronenborn ◽  
C. M. Clore ◽  
A. C. Steven
Keyword(s):  
Solution Structure ◽  
Carbon Films ◽  
Uranyl Acetate ◽  
Protein Solutions ◽  
Human Macrophage ◽  
High Concentration ◽  
Nmr Studies ◽  
Inflammatory Protein ◽  
Extensive Class ◽  
Negative Staining Electron Microscopy

Human macrophage inflammatory protein 1β (hMIP-1β), also known as Act-2, is a small protein that belongs to an extensive class of chemotactic cytokines. The high resolution solution structure of hMIP-1β has recently been determined by multidimensional NMR spectroscopy, revealing a dimeric molecule of 2 x 8 kDa. The fold of the monomer is very similar to that of the related cytokine, interleukin-8, which was solved previously. However, the mode of association whereby two monomers form a dimer is quite different. To maintain a sufficiently high concentration of soluble protein for NMR studies, hMIP-1β was dissolved at a pH of 2.5. During solubility trials, it was noticed that the hMIP-1β-containing solutions became birefringent at higher pHs, suggesting the formation of ordered light-scattering structures. We have investigated this phenomenon by negative staining electron microscopy.hMIP-1β was obtained from an expression vector, extracted from inclusion bodies, and purified as described. Protein solutions at 0.7 mg/ml, 100 mM NaCl, 20mM Na citrate, pH2.3, were dialyzed against 20mM ionic strength buffers at pH 4.5, 5.0 and 5.5 (sodium citrate), and pH 6.0 and 7.0 (sodium phosphate) at 4°C. Samples were withdrawn at various time-points, applied to glow-discharged carbon films, stained briefly (5s) with uranyl acetate, and examined in a Philips EM400RT microscope.

scholarly journals Human Macrophage Inflammatory Protein 3α: Protein and Peptide Nuclear Magnetic Resonance Solution Structures, Dimerization, Dynamics, and Anti-Infective Properties

2007 ◽  
Vol 52 (3) ◽  
pp. 883-894 ◽  
Author(s):  
David I. Chan ◽  
Howard N. Hunter ◽  
Brian F. Tack ◽  
Hans J. Vogel
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Crystal Structures ◽  
Solution Structure ◽  
Proton Nuclear Magnetic Resonance ◽  
Human Macrophage ◽  
Inflammatory Protein ◽  
Α Helix ◽  
Macrophage Inflammatory Protein ◽  
Nuclear Magnetic

ABSTRACT Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70-amino-acid chemokine which exclusively binds to chemokine receptor 6. In addition, the protein also has direct antimicrobial, antifungal, and antiviral activities. The solution structure of MIP-3α was solved by the use of two-dimensional homonuclear proton nuclear magnetic resonance (NMR). The structure reveals the characteristic chemokine fold, with three antiparallel β strands followed by a C-terminal α helix. In contrast to the crystal structures of MIP-3α, the solution structure was found to be monomeric. Another difference between the NMR and crystal structures lies in the angle of the α helix with respect to the β strands, which measure 69 and ∼56.5° in the two structures, respectively. NMR diffusion and pH titration studies revealed a distinct tendency for MIP-3α to form dimers at neutral pH and monomers at lower pH, dependent on the protonation state of His40. Molecular dynamics simulations of both the monomeric and the dimeric forms of MIP-3α supported the notion that the chemokine undergoes a change in helix angle upon dimerization and also highlighted the important hydrophobic and hydrogen bonding contacts made by His40 in the dimer interface. Moreover, a constrained N terminus and a smaller binding groove were observed in dimeric MIP-3α simulations, which could explain why monomeric MIP-3α may be more adept at receptor binding and activation. The solution structure of a synthetic peptide consisting of the last 20 residues of MIP-3α displayed a highly amphipathic α helix, reminiscent of various antimicrobial peptides. Antimicrobial assays with this peptide revealed strong and moderate bactericidal activities against Escherichia coli and Staphylococcus aureus, respectively. This confirms that the C-terminal α-helical region of MIP-3α plays a significant part in its broad anti-infective activity.

Insights Into Crowding Effects on Protein Stability From a Coarse-Grained Model

2009 ◽  
Vol 131 (7) ◽  
Author(s):  
Vincent K. Shen ◽  
Jason K. Cheung ◽  
Jeffrey R. Errington ◽  
Thomas M. Truskett
Keyword(s):  
Protein Stability ◽  
Coarse Grained ◽  
Protein Solutions ◽  
Native State ◽  
High Concentration ◽  
Coarse Grained Model ◽  
Excluded Volume Effects ◽  
Thermodynamic Phase ◽  
Broad Interest ◽  
Liquid Liquid Phase Separation

Proteins aggregate and precipitate from high concentration solutions in a wide variety of problems of natural and technological interest. Consequently, there is a broad interest in developing new ways to model the thermodynamic and kinetic aspects of protein stability in these crowded cellular or solution environments. We use a coarse-grained modeling approach to study the effects of different crowding agents on the conformational equilibria of proteins and the thermodynamic phase behavior of their solutions. At low to moderate protein concentrations, we find that crowding species can either stabilize or destabilize the native state, depending on the strength of their attractive interaction with the proteins. At high protein concentrations, crowders tend to stabilize the native state due to excluded volume effects, irrespective of the strength of the crowder-protein attraction. Crowding agents reduce the tendency of protein solutions to undergo a liquid-liquid phase separation driven by strong protein-protein attractions. The aforementioned equilibrium trends represent, to our knowledge, the first simulation predictions for how the properties of crowding species impact the global thermodynamic stability of proteins and their solutions.

scholarly journals Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes.

1993 ◽  
Vol 177 (6) ◽  
pp. 1821-1826 ◽  
Author(s):  
T J Schall ◽  
K Bacon ◽  
R D Camp ◽  
J W Kaspari ◽  
D V Goeddel
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cytotoxic T Cells ◽  
Human Macrophage ◽  
Immune Effector ◽  
Effector Cells ◽  
Inflammatory Protein ◽  
And Migration ◽  
Macrophage Inflammatory Protein

Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration-dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.

scholarly journals Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor.

1993 ◽  
Vol 177 (5) ◽  
pp. 1421-1427 ◽  
Author(s):  
J L Gao ◽  
D B Kuhns ◽  
H L Tiffany ◽  
D McDermott ◽  
X Li ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Functional Expression ◽  
Amino Acid Identity ◽  
Human Macrophage ◽  
Hematopoietic Stem ◽  
Reading Frame ◽  
Inflammatory Protein ◽  
Common Receptor ◽  
Immature Myeloid Cells ◽  
Macrophage Inflammatory Protein

The chemokine beta family is comprised of at least six distinct cytokines that regulate trafficking of phagocytes and lymphocytes in mammalian species; at least one of these, macrophage inflammatory protein 1 alpha (MIP-1 alpha), also regulates the growth of hematopoietic stem cells. We now show that MIP-1 alpha and the related beta chemokine, RANTES, induce transient alterations in intracellular Ca2+ concentration in polymorphonuclear leukocytes that can be reciprocally and specifically desensitized, suggesting a common receptor. Moreover, we have now cloned both the cDNA and the gene for this receptor, functionally expressed the receptor in Xenopus oocytes, and mapped the gene to human chromosome 3p21. Transcripts for the receptor were found in mature and immature myeloid cells as well as B cells. The receptor is a member of the G protein-coupled receptor superfamily. It has approximately 33% amino acid identity with receptors for the alpha chemokine, interleukin 8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.

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