The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein.

Essentials Recombinant factor VIII (rFVIII) Fc fusion protein has a 1.5-fold longer half-life than rFVIII. Five orthogonal methods were used to characterize the structure of rFVIIIFc compared to rFVIII. The C-terminal Fc fusion does not perturb the structure of FVIII in rFVIIIFc. The FVIII and Fc components of rFVIIIFc are flexibly tethered and functionally independent. SUMMARY: Background Fusion of the human IgG1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. Objective To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. Methods rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), and electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). Results HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Conclusions The FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.

Molecular subtype and tumor characteristics of breast cancer metastases as assessed by gene expression significantly influence patient post-relapse survival.

BACKGROUND: We and others have recently shown that tumor characteristics are altered throughout tumor progression. These findings emphasize the need for re-examination of tumor characteristics at relapse and have led to recommendations from ESMO and the Swedish Breast Cancer group. Here, we aim to determine whether tumor characteristics and molecular subtypes in breast cancer metastases confer clinically relevant prognostic information for patients. PATIENTS AND METHODS: The translational aspect of the Swedish multicenter randomized trial called TEX included 111 patients with at least one biopsy from a morphologically confirmed locoregional or distant breast cancer metastasis diagnosed from December 2002 until June 2007. All patients had detailed clinical information, complete follow-up, and metastasis gene expression information (Affymetrix array GPL10379). We assessed the previously published gene expression modules describing biological processes [proliferation, apoptosis, human epidermal receptor 2 (HER2) and estrogen (ER) signaling, tumor invasion, immune response, and angiogenesis] and pathways (Ras, MAPK, PTEN, AKT-MTOR, PI3KCA, IGF1, Src, Myc, E2F3, and ß-catenin) and the intrinsic subtypes (PAM50). Furthermore, by contrasting genes expressed in the metastases in relation to survival, we derived a poor metastasis survival signature. RESULTS: A significant reduction in post-relapse breast cancer-specific survival was associated with low-ER receptor signaling and apoptosis gene module scores, and high AKT-MTOR, Ras, and ß-catenin module scores. Similarly, intrinsic subtyping of the metastases provided statistically significant post-relapse survival information with the worst survival outcome in the basal-like [hazard ratio (HR) 3.7; 95% confidence interval (CI) 1.3-10.9] and HER2-enriched (HR 4.4; 95% CI 1.5-12.8) subtypes compared with the luminal A subtype. Overall, 25% of the metastases were basal-like, 32% HER2-enriched, 10% luminal A, 28% luminal B, and 5% normal-like. CONCLUSIONS: We show that tumor characteristics and molecular subtypes of breast cancer metastases significantly influence post-relapse patient survival, emphasizing that molecular investigations at relapse provide prognostic and clinically relevant information. CLINICALTRIALS.GOV: This is the translational part of the Swedish multicenter and randomized trial TEX, clinicaltrials.gov identifier nct01433614 (http://www.clinicaltrials.gov/ct2/show/nct01433614).

Serum thymidine kinase activity compared with CA 15-3 in locally advanced and metastatic breast cancer within a randomized trial.

The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum™ assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15 months p = 0.02) and OS (21 vs. 38 months, p < 0.0001), respectively. After adjustment for age, metastatic site and study treatment TK1 showed a trend as predictor of PFS (p = 0.059) and was an independent prognostic factor for OS, (HR 1.81, 95 % confidence interval (CI) 1.26-2.61, p = 0.001). There was a trend of shortened OS for women with high CA 15-3 (p = 0.054) in univariate analysis, but not after adjustment for the above mentioned covariates. Both TK1 (p = 0.0011) and CA 15-3 (p = 0.0004) predicted response to treatment. There were statistically different distributions of TK1 and CA 15-3 in relation to the site of metastases. TK1 activity measured by DiviTum™ predicted therapy response, PFS and OS in loco regional inoperable or disseminated breast cancer. These results suggest that this factor is a useful serum marker. In the present material, a prognostic value of CA 15-3 could not be proven.

Individually tailored treatment with epirubicin and paclitaxel with or without capecitabine as first-line chemotherapy in metastatic breast cancer: a randomized multicenter trial.

Anthracyclines and taxanes are active cytotoxic drugs in the treatment of early metastatic breast cancer. It is yet unclear whether addition of capecitabine to the combination of these drugs improves the treatment outcome. Patients with advanced breast cancer were randomized to first-line chemotherapy with a combination of epirubicin (Farmorubicin(®)) and paclitaxel (Taxol(®)) alone (ET) or in combination with capecitabine (Xeloda(®), TEX). Starting doses for ET were epirubicin 75 mg/m(2) plus paclitaxel 175 mg/m(2), and for TEX epirubicin 75 mg/m(2), paclitaxel 155 mg/m(2), and capecitabine 825 mg/m(2) BID for 14 days. Subsequently, doses were tailored related to side effects. Primary endpoint was progression-free survival (PFS); secondary endpoints were overall survival (OS), time to treatment failure (TTF), objective response (OR), safety and quality of life (QoL). 287 patients were randomized, 143 to ET and 144 to TEX. Median PFS was 10.8 months for patients treated with ET, and 12.4 months for those treated with TEX (HR 0.84, 95% CI 0.65-1.07, P = 0.16); median OS was 26.0 months for women in the ET versus 29.7 months in the TEX arm (HR 0.84, 95% CI 0.63-1.11, P = 0.22). OR was achieved in 44.8% (ET) and 54.2% (TEX), respectively (χ(2) 3.66, P = 0.16). TTF was significantly longer for patients treated with TEX, 6.0 months, versus 5.2 months following ET (HR 0.73, 95% CI 0.58-0.93, P = 0.009). Severe hematological side effects related to epirubicin and paclitaxel were evenly distributed between the treatment arms, mucositis, diarrhea, and Hand-Foot syndrome were significantly more frequent in the TEX arm. Toxicity-adjusted treatment with ET and TEX showed similar efficacy in terms of PFS, OS, and OR. In this trial with limited power, the addition of capecitabine to epirubicin and paclitaxel as first-line treatment did not translate into clinically relevant improvement of the outcome.

TGF-alpha and ErbB2 production in synovial joint tissue: increased expression in arthritic joints.

OBJECTIVE: Cell types present in synovial joint tissues and during synovitis are known to produce epidermal growth factor receptor (EGFR)/ErbB-1/HER-1 and the potent EGFR-ligand transforming growth factor-alpha (TGF-alpha) in vitro. Concomitant expression of TGF-alpha, EGFR, and ErbB2 gives a strong proliferative drive in vitro and in vivo. However, the presence of TGF-alpha and members of the EGFR/EGFR-ligand family has not been thoroughly investigated in joint tissue in vivo. We aimed to determine whether TGF-alpha, EGFR, and ErbB2 are present in human synovial joints, especially during rheumatoid arthritis (RA). METHODS: TGF-alpha protein was immunodetected in knee synovial fluid (SF) collected from 23 RA patients, eight patients with other arthritic conditions, two osteoarthritis (OA) patients, and six post-traumatic patients (control). TGF-alpha mRNA and TGF-alpha, ErbB2, EGFR, and CD68 immunoreactivity were detected in knee synovial biopsies (6 RA/2 OA/6 control) using in situ hybridization and immunohistochemistry. TGF-alpha mRNA was determined in SF cells by reverse transcription polymerase chain reaction (RT-PCR) and/or the Northern blot technique. RESULTS: TGF-alpha protein was found in the synovial membrane (SM) and in the majority of SF samples. TGF-alpha levels were significantly higher (p < 0.001) in SF of RA patients than controls, TGF-alpha protein and mRNA were increased and more widespread in SM of RA patients. In addition, white blood cells collected from RA SF expressed TGF-alpha mRNA. Immunoreactivity for ErbB2 was found in SM and was more widespread in RA patients than in controls. CONCLUSION: The presence of TGF-alpha in normal SF and SM may indicate a physiological maintenance function. The increased expression of TGF-alpha and ErbB2 in RA SF and SM may give rise to an abnormal growth pattern, contributing to inflammatory synovial hyperplasia.

Structure of the Sec23p/24p and Sec13p/31p complexes of COPII.

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes. The three-dimensional reconstruction of Sec23p/24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p/31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28-30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPII-coated vesicles, we present a model for COPII coat organization.

Genes that co-cluster with estrogen receptor alpha in microarray analysis of breast biopsies.

The estrogen receptor plays a critical role in the pathogenesis and clinical behavior of breast cancer. To better understand the molecular basis of estrogen-dependent forms of this disease we studied gene expression profiles from 53 primary breast cancer biopsies. Gene expression data for more than 7000 genes were generated from each tumor sample with oligo microarrays. A standard correlation-clustering algorithm identified 18 genes that co-clustered with estrogen receptor alpha. Eleven of these genes had previously been associated with estrogen regulation or breast tumorigenesis including trefoil factor 1 and estrogen regulated LIV-1. Additional study of these 18 genes may further delineate the role of estrogen receptor in breast cancer, generate new predictive biomarkers for response to endocrine therapies and identify novel therapeutic targets.

Interleukin-6 enhances transforming growth factor-alpha mRNA expression in macrophage-like human monocytoid (U-937-1) cells.

We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-alpha) and that the steady-state levels of TGF-alpha mRNA as well as TGF-alpha protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF-alpha expression in keratinocytes. In the present study we investigated whether TGF-alpha expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation. U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF-alpha mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF-alpha gene was accompanied by release of TGF-alpha protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF-alpha as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF-alpha gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF-alpha protein release or pro-TGF-alpha surface expression. We conclude that since IL-6 causes increased steady-state levels of TGF-alpha mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA.

Structural determinants of water permeation through aquaporin-1.

Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

Is morphoea caused by Borrelia burgdorferi? A review.

The aetiology of morphoea and lichen sclerosus et atrophicus is still unknown. Since the detection of Borrelia burgdorferi (B. burgdorferi) as the causative agent of Lyme disease, there has been debate about a possible association between B. burgdorferi and morphoea. Initial serological and cultural studies showed controversial results. The introduction of polymerase chain reaction (PCR) initially suggested an association between B. burgdorferi and morphoea. We reviewed the literature on B. burgdorferi (specific serology, immunohistology, culture, lymphocyte stimulation and DNA detection by PCR) since 1983, using Medline and Current Contents. Histological and immunohistological detection of B. burgdorferi was reported in 0-40% (20 of 82) of the cases with morphoea and in 46-50% (17 of 36) of the cases with lichen sclerosus et atrophicus. Cultivation of spirochetes from lesional skin succeeded in five patients (five of 68) with morphoea, but failed in patients with lichen sclerosus et atrophicus. In Europe and Asia, serological detection of antibodies against B. burgdorferi was described in 0-60% (138 of 609) of patients with morphoea and in 19% (six of 32) in the U.S.A. For lichen sclerosus et atrophicus 0-25% of the published cases (three of 23) in Europe and Asia were seropositive. DNA from B. burgdorferi was detected by PCR in 0-100% (17 of 82) of the tissues of patients with morphoea in Europe and Asia, but not a single case among 98 patients was reported to be positive from the U. S.A. In Europe and Asia, borrelial DNA was detected in 0-100% (nine of 28) of the cases with lichen sclerosus et atrophicus, whereas in the U.S.A. none of 48 patients was positive. There are two possible explanations for these contradictory findings: the most likely is that B. burgdorferi is not a causative agent for morphoea. Another possible explanation could be that a subset of morphoea is caused by a special subspecies of B. burgdorferi that is present in Europe and Asia but does not occur in the U.S.A.

Gandhian principles in social work practice: ethics revisited.

Social work as an expression of culture is a highly value-laden activity. The emergence of many new ethical issues resulting from technological and scientific advancements suggests a need for greater attention to values and ethics. In this article the authors argue that the thought of Mahatma Gandhi, as revealed in his social activism, is relevant to social work ethics and a resource for its ethical enrichment. Principles such as seeking truth through service to others, individual self-development, nonviolent social action, and material simplicity could enhance the current NASW Code of Ethics.

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis.

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.

Human melanoma progression in skin reconstructs : biological significance of bFGF.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

The structure of aquaporin-1 at 4.5-A resolution reveals short alpha-helices in the center of the monomer.

Aquaporin-1 is a water channel found in mammalian red blood cells that is responsible for high water permeability of its membrane. Our electron crystallographic analysis of the three-dimensional structure of aquaporin-1 at 4.5-A resolution confirms the previous finding that each subunit consists of a right-handed bundle of six highly tilted transmembrane helices that surround a central X-shaped structure. In our new potential map, the rod-like densities for the transmembrane helices show helically arranged protrusions, indicating the positions of side chains. Thus, in addition to the six transmembrane helices, observation of helically arranged side-chain densities allowed the identification of two short alpha-helices representing the two branches of the central X-shaped structure that extend to the extracellular and cytoplasmic membrane surfaces. The other two branches are believed to be loops connecting the short alpha-helix to a neighboring transmembrane helix. A pore found close to the center of the aquaporin-1 monomer is suggested to be the course of water flow with implications for the water selectivity.

Effects of human platelet-derived growth factor-AB on sarcoma growth in vitro and in vivo.

Platelet-derived growth factor (PDGF) has been proposed to play an important role in the growth of tumors. In order to study the effects of PDGF-AB on tumor growth in vivo, sarcoma-bearing mice were treated with PDGF-AB. The tumors, a malignant fibrous histiocytoma and an osteosarcoma, had functional PDGF receptors in vitro, as demonstrated by stimulation of PDGF-AB using a [3H]thymidine incorporation assay. Immunohistochemistry also revealed that both sarcoma xenografts expressed PDGF receptors. The tumor-bearing mice were given human PDGF-AB for 14 days, either continuously by an intraperitoneally placed mini-osmotic pump, or by daily injections. No effects on tumor growth in vivo were observed, as measured by tumor volume, autoradiography or cell cycle distribution. The histological appearance and ploidy of the tumors remained unaltered. The results indicate that, although the tumor cells are stimulated by PDGF-AB in vitro, the in vivo milieu or tumor growth pattern may render the tumors less susceptible to exogenously administered PDGF-AB in vivo.

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Suramin blocks growth-stimulatory effects of platelet-derived growth factor on malignant fibrous histiocytomas in vitro.

The pattern of susceptibility of malignant cells to the cytostatic drug suramin is not fully clarified. Therefore, in the present paper we have assessed the effects of suramin on the growth of eight cell lines derived from human malignant fibrous histiocytomas, by measuring DNA synthesis. The effect of suramin (10-200 microg/ml) on cells either unstimulated, or stimulated with platelet-derived growth factor (PDGF)-AA (10 ng/ml), PDGF-BB (10 ng/ml) or 10% fetal calf serum was studied. Four out of five cell lines unable to thrive without external growth factors showed growth inhibition by suramin. The two cell lines able to grow under serum-free conditions were unaffected by high-dose suramin. The exposure to suramin, at 200 microg/ml, abolished the growth stimulation caused by PDGF-AA and -BB. In contrast, a low dose of suramin (50 microg/ml), with or without PDGF, caused growth-stimulating effects in some cell lines. Our results indicate that high doses of suramin inhibit growth of malignant fibrous histiocytomas in vitro and that suramin exerts its growth-inhibitory effects on cells dependent on external growth factors. Low-dose treatment with suramin, however, may instead promote growth in both serum-dependent and -independent tumor cell lines.

Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A.

Three photosynthetic complexes, light-harvesting complex 2 (LH2), light-harvesting complex 1 (LH1), and the reaction centre-light-harvesting complex 1 photounit (RC-LH1), were purified from a single species of a purple bacterium, Rhodobacter sphaeroides, and reconstituted into two-dimensional (2-D) crystals. Vesicular 2-D crystals of LH1 and RC-LH1 were imaged in negative stain and projection maps at 25 A resolution were produced. The rings formed by LH1 have approximately the same mean diameter as the LH1 rings from Rhodospirillum rubrum ( approximately 90 A) and therefore are likely to be composed of 15 to 17 alphabeta subunits. In the projection map calculated from the RC-LH1 2-D crystals, the reaction centre is represented by an additional density in the centre of the ring formed by the LH1 subunits. The marked improvement of shape and fine structure after a rotational pre-alignment of the RC-LH1 unit cells before averaging strongly suggests that the RC is not in a unique orientation within the LH1 rings. Tubular crystals of LH2 showed a high degree of order and allowed calculation of a projection map at 6 A resolution from glucose-embedded specimens. The projection structure shows a ring of nine alphabeta subunits. Variation of the alpha-helical projection densities suggests that the 9-fold symmetry axis is tilted with respect to the membrane normal.

2D crystallization of membrane proteins: rationales and examples.

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy. While reconstitution of membrane proteins with lipids is readily achieved, the mechanisms for crystal formation during or after reconstitution are not well understood. The nature of the detergent and lipid as well as pH and counter-ions is known to influence the crystal type and quality. Protein-protein interactions may also promote crystal stacking and aggregation of the sheet-like crystals, posing problems in data collection. Although highly promising, the number of well-studied examples is still too small to draw conclusions that would be applicable to any membrane protein of interest. Here we discuss parameters influencing the outcome of two-dimensional crystallization trials using prominent examples of channel protein crystals and highlight areas where further improvements to crystallization protocols can be made.