Water-inserted alpha-helical segments implicate reverse turns as folding intermediates

Information relevant to the folding and unfolding of alpha helices has been extracted from an analysis of protein structures. The alpha helices in protein crystal structures have been found to be hydrated, either externally by a water molecule hydrogen bonding to the backbone carbonyl oxygen atom, or internally by inserting into the helix hydrogen bond and forming a hydrogen-bonded bridge between the backbone carbonyl oxygen and the amide nitrogen atoms. The water-inserted alpha-helical segments display a variety of reverse-turn conformations, such as type III, type II, type I, and opened out, that can be considered as folding intermediates that are trapped in the folding-unfolding process of alpha helices. Since the alpha helix, most turns, and the extended beta strand occupy contiguous regions in the conformational space of phi, psi dihedral angles, a plausible pathway can be proposed for the folding-unfolding process of alpha helices in aqueous solution.     

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.     

Three-dimensional structure of recombinant human interferon-gamma

The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.     

Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae

The membrane rotor ring from the vacuolar-type (V-type) sodium ion-pumping adenosine triphosphatase (Na+-ATPase) from Enterococcus hirae consists of 10 NtpK subunits, which are homologs of the 16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F1Fo- or F-ATPases, respectively. Each NtpK subunit has four transmembrane alpha helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.     

Seeing the herpesvirus capsid at 8.5 A

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative alpha helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.     

Direct observation of chaperone-induced changes in a protein folding pathway

How chaperone interactions affect protein folding pathways is a central problem in biology. With the use of optical tweezers and all-atom molecular dynamics simulations, we studied the effect of chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) at the single-molecule level. In the absence of SecB, we find that the MBP polypeptide first collapses into a molten globulelike compacted state and then folds into a stable core structure onto which several alpha helices are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the core structure but have no detectable effect on the folding of the external alpha helices. It appears that SecB only binds to the extended or molten globulelike structure and retains MBP in this latter state. Thus during MBP translocation, no energy is required to disrupt stable tertiary interactions.     

Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.     

Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E

Human apolipoprotein E, a blood plasma protein, mediates the transport and uptake of cholesterol and lipid by way of its high affinity interaction with different cellular receptors, including the low-density lipoprotein (LDL) receptor. The three-dimensional structure of the LDL receptor-binding domain of apoE has been determined at 2.5 angstrom resolution by x-ray crystallography. The protein forms an unusually elongated (65 angstroms) four-helix bundle, with the helices apparently stabilized by a tightly packed hydrophobic core that includes leucine zipper-type interactions and by numerous salt bridges on the mostly charged surface. Basic amino acids important for LDL receptor binding are clustered into a surface patch on one long helix. This structure provides the basis for understanding the behavior of naturally occurring mutants that can lead to atherosclerosis.     

Reexamination of the Three-Dimensional Structure of the Small Subunit of RuBisCo from Higher Plants

The structure of L(8)S(8) RuBisCo (where L is the large subunit and S is the small subunit) from spinach has been determined to a resolution of 2.8 ?ngstrom by using fourfold averaging of an isomorphous electron density map based on three heavy-atom derivatives. The structure of the S subunit is different from that previously reported for the tobacco S subunit in spite of 75 percent sequence identity. The elements of secondary structure, four antiparallel beta strands and two alpha helices, are the same, but the topology and direction of the polypeptide chain through these elements differ completely. One of these models is clearly wrong. The spinach model has hydrophobic residues in the core between the alpha helices and beta sheet as well as conserved residues in the subunit interactions. The deletion of residues 49 to 62 that is present in the Anabaena sequence removes a loop region in the spinach model. The positions of three mercury atoms in the heavy-atom derivatives agree with the assignment of side chains in the spinach structure.     

杀伤细胞免疫球蛋白样受体在输血中的作用

目的 初步探讨杀伤细胞免疫球蛋白样受体在输血中的作用.方法 用免疫磁珠法分离正常人外周血NK细胞,再用PCR-SSP方法进行KIR分型.将分型的NK细胞与已知HLA分型的全血混合,37℃培养0、6、12、24h,ELISA法检测IL-2、IL-4、前列腺素E2(PGE2)、TNF-α、TNF-β、IFN-γ、粒细胞-巨...     

Amino acid preferences for specific locations at the ends of alpha helices

A definition based on alpha-carbon positions and a sample of 215 alpha helices from 45 different globular protein structures were used to tabulate amino acid preferences for 16 individual positions relative to the helix ends. The interface residue, which is half in and half out of the helix, is called the N-cap or C-cap, whichever is appropriate. The results confirm earlier observations, such as asymmetrical charge distributions in the first and last helical turn, but several new, sharp preferences are found as well. The most striking of these are a 3.5:1 preference for Asn at the N-cap position, and a preference of 2.6:1 for Pro at N-cap + 1. The C-cap position is overwhelmingly dominated by Gly, which ends 34 percent of the helices. Hydrophobic residues peak at positions N-cap + 4 and C-cap - 4.     

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.     

Helix signals in proteins

The alpha helix, first proposed by Pauling and co-workers, is a hallmark of protein structure, and much effort has been directed toward understanding which sequences can form helices. The helix hypothesis, introduced here, provides a tentative answer to this question. The hypothesis states that a necessary condition for helix formation is the presence of residues flanking the helix termini whose side chains can form hydrogen bonds with the initial four-helix greater than N-H groups and final four-helix greater than C-O groups; these eight groups would otherwise lack intrahelical partners. This simple hypothesis implies the existence of a stereochemical code in which certain sequences have the hydrogen-bonding capacity to function as helix boundaries and thereby enable the helix to form autonomously. The three-dimensional structure of a protein is a consequence of the genetic code, but the rules relating sequence to structure are still unknown. The ensuing analysis supports the idea that a stereochemical code for the alpha helix resides in its boundary residues.     

Partial symmetrization of the photosynthetic reaction center

The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that performs the primary charge separation event of photosynthesis, thereby converting light to chemical energy. The RC pigments are bound primarily by two homologous peptides, the L and M subunits, each containing five transmembrane helices. These alpha helices and pigments are arranged in an approximate C2 symmetry and form two possible electron transfer pathways. Only one of these pathways is actually used. In an attempt to identify nonhomologous residues that are responsible for functional differences between the two branches, homologous helical regions that interact extensively with the pigments were genetically symmetrized (that is, exchanged). For example, replacement of the fourth transmembrane helix (D helix) in the M subunit with the homologous helix from the L subunit yields photosynthetically inactive RCs lacking a critical photoactive pigment. Photosynthetic revertants have been isolated in which single amino acid substitutions (intragenic suppressors) compensate for this partial symmetrization.     

Tertiary structure of plant RuBisCO: domains and their contacts

The three-dimensional structure of ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO), has been determined at 2.6 A resolution. This enzyme initiates photosynthesis by combining carbon dioxide with ribulose bisphosphate to form two molecules of 3-phosphoglycerate. In plants, RuBisCO is built from eight large (L) and eight small (S) polypeptide chains, or subunits. Both S chains and the NH2-terminal domain (N) of L are antiparallel beta, "open-face-sandwich" domains with four-stranded beta sheets and flanking alpha helices. The main domain (B) of L is an alpha/beta barrel containing most of the catalytic residues. The active site is in a pocket at the opening of the barrel that is partly covered by the N domain of a neighboring L chain. The domain contacts of the molecule and its conserved residues are discussed in terms of this structure.     

Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's

Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.     

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.     

A G protein mutant that inhibits thrombin and purinergic receptor activation of phospholipase A2

The stimulation of phospholipase A2 by thrombin and type 2 (P2)-purinergic receptor agonists in Chinese hamster ovary cells is mediated by the G protein Gi. To delineate alpha chain regulatory regions responsible for control of phospholipase A2, chimeric cDNAs were constructed in which different lengths of the alpha subunit of Gs (alpha s) were replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). When a carboxyl-terminal chimera alpha s-i(38), which has the last 38 amino acids of alpha s substituted with the last 36 residues of alpha i2, was expressed in Chinese hamster ovary cells, the receptor-stimulated phospholipase A2 activity was inhibited, although the chimera could still activate adenylyl cyclase. Thus, alpha s-i(38) is an active alpha s, but also a dominant negative alpha i molecule, indicating that the last 36 amino acids of alpha i2 are a critical domain for G protein regulation of phospholipase A2 activity.