FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079347968> ?p ?o ?g. }

• W2079347968 endingPage "17196" @default.
• W2079347968 startingPage "17190" @default.
• W2079347968 abstract "The von-Hippel Lindau tumor suppressor protein (pVHL) is conserved throughout evolution, as its homologues are found in organisms ranging from mammals to the Drosophila melanogaster and Anopheles gambiae insects and the Caenorhabditis elegans nematode. Although the physiological role of pVHL is not fully understood, it has been shown to interact with a large number of unrelated proteins and was suggested to play a role in protein degradation as an E3 ubiquitin ligase component in the ubiquitin pathway. To gain insight into the molecular basis of pVHL activity, we analyzed its folding and stability in solution under physiologically relevant conditions. Dynamic light-scattering and gel filtration chromatography of the purified pVHL clearly indicated that the Stokes radius of the protein is larger than what would be expected from its crystal structure. However, under these conditions, the protein shows a clear secondary structure as determined by far-UV circular dichroism. Yet, the near-UV CD experiments show an absence of a tertiary structure. Upon the addition of urea, even at very low concentrations, the protein unfolds in a non-reversible manner, leading to the formation of amorphous aggregates. Furthermore, a large increase in fluorescence (>50-fold) is observed upon the addition of pVHL into a solution containing 8-anilino-1-naphthalene sulfonic acid. We therefore conclude that, under native conditions, the non-bound pVHL has a molten globule configuration with marginal stability. Although molten globular structures can be induced in many proteins under extreme conditions, this is one of the few reported cases of such a structure under the physiological conditions of pH, ionic strength, and temperature. The significance of the pVHL structural properties is being discussed in the context of its physiological activities. The von-Hippel Lindau tumor suppressor protein (pVHL) is conserved throughout evolution, as its homologues are found in organisms ranging from mammals to the Drosophila melanogaster and Anopheles gambiae insects and the Caenorhabditis elegans nematode. Although the physiological role of pVHL is not fully understood, it has been shown to interact with a large number of unrelated proteins and was suggested to play a role in protein degradation as an E3 ubiquitin ligase component in the ubiquitin pathway. To gain insight into the molecular basis of pVHL activity, we analyzed its folding and stability in solution under physiologically relevant conditions. Dynamic light-scattering and gel filtration chromatography of the purified pVHL clearly indicated that the Stokes radius of the protein is larger than what would be expected from its crystal structure. However, under these conditions, the protein shows a clear secondary structure as determined by far-UV circular dichroism. Yet, the near-UV CD experiments show an absence of a tertiary structure. Upon the addition of urea, even at very low concentrations, the protein unfolds in a non-reversible manner, leading to the formation of amorphous aggregates. Furthermore, a large increase in fluorescence (>50-fold) is observed upon the addition of pVHL into a solution containing 8-anilino-1-naphthalene sulfonic acid. We therefore conclude that, under native conditions, the non-bound pVHL has a molten globule configuration with marginal stability. Although molten globular structures can be induced in many proteins under extreme conditions, this is one of the few reported cases of such a structure under the physiological conditions of pH, ionic strength, and temperature. The significance of the pVHL structural properties is being discussed in the context of its physiological activities. Mutations in the von Hippel-Lindau (VHL) 1The abbreviations used are: VHL, von-Hippel Lindau; pVHL, VHL tumor suppressor protein; ANSA, 8-anilino-1-naphthalene sulfonic acid; VCB, VHL-Elongin C-Elongin B; VCB-CR, VHL-Elongin C-Elongin B-Cul2-Rbx1; HIF, hypoxia-inducible factor; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Mw, weight average Mr; RS, Stokes radius; RH, hydrodynamic radius. tumor suppressor protein (pVHL) are associated with the hereditary VHL syndrome. This disease is characterized by a predisposition to develop retinal angiomas, cerebellar and spinal hemangioblastomas, renal cell carcinomas, pheochromocytomas, pancreatic adenomas and islet cell tumors, epididymal cystadenomas, and endolymphatic sac tumors of the inner ear (1Maher E.R. Kaelin Jr., W.G. Medicine (Baltimore). 1997; 76: 381-391Crossref PubMed Scopus (434) Google Scholar). The inactivation of the VHL protein occurs at the early stages of the pathogenesis of kidney lesions and clear cell renal carcinoma and is considered to be the most frequent genetic event at the onset of human kidney cancer (2Kaelin Jr., W.G. Maher E.R. Trends Genet. 1998; 14: 423-426Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 3Ohh M. Kaelin Jr., W.G. Mol. Med. Today. 1999; 5: 257-263Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The product of the VHL gene is a 213-amino acid protein (4Iliopoulos O. Ohh M. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11661-11666Crossref PubMed Scopus (209) Google Scholar). A second form of pVHL is generated by translation initiation at an internal methionine, located at residue 54, with a molecular mass of 19 kDa (4Iliopoulos O. Ohh M. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11661-11666Crossref PubMed Scopus (209) Google Scholar). The structure of the 19-kDa pVHL protein was determined by x-ray crystallography in a ternary complex with Elongin C and Elongin B (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar). Another recently elucidated crystal structure of this complex, which interacts with a 20-residue destruction sequence of HIF-1α and contains a functional hydroxyproline, was recently elucidated (6Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (590) Google Scholar). The crystal structures show that the pVHL molecule consists of two domains, α and β. Both domains are connected by two short polypeptide linkers and a polar interface that is stabilized by a hydrogen bond network. The β-domain of pVHL consists of a seven-stranded β-sandwich (residues 63–154) and an α-helix (residues 193–204) that packs against one of the β-sheets through hydrophobic interactions. The α-domain of pVHL (residues 155–192) consists of three α-helices (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar). Still, no unbound crystal structures or structures of pVHL in solution are available. Like other tumor suppressors, the pVHL protein has a regulatory function in many cellular pathways, and it is suggested that many different factors interact with the pVHL protein in direct or indirect forms (7Kaelin Jr., W.G. Nat. Rev. Cancer. 2002; 2: 673-682Crossref PubMed Scopus (698) Google Scholar). The pVHL protein is suggested to have a role in the regulation of transcription of many downstream elements. Biochemical studies have revealed that pVHL forms a ternary complex (VCB) with Elongin C and Elongin B proteins via a similar binding site, as exists in Elongin A (8Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin Jr., W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (576) Google Scholar, 9Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (293) Google Scholar, 10Conaway J.W. Kamura T. Conaway R.C. Biochim. Biophys. Acta. 1998; 1377: M49-54PubMed Google Scholar). This interaction could indicate that one of the pVHL tumor suppressor roles is inhibition of elongation due to competition with Elongin A in the complex formation (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar, 8Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin Jr., W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (576) Google Scholar, 9Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (293) Google Scholar, 10Conaway J.W. Kamura T. Conaway R.C. Biochim. Biophys. Acta. 1998; 1377: M49-54PubMed Google Scholar, 11Duan D.R. Pause A. Burgess W.H. Aso T. Chen D.Y. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (513) Google Scholar). Furthermore, it has been demonstrated that pVHL binds directly to the transcription factor sp1 in vitro, hence suggesting that pVHL could be a regulator of the transcription of vascular endothelial growth factor (VEGF) mRNA (12Mukhopadhyay D. Knebelmann B. Cohen H.T. Ananth S. Sukhatme V.P. Mol. Cell. Biol. 1997; 17: 5629-5639Crossref PubMed Scopus (309) Google Scholar). Subsequently, it was demonstrated that pVHL binds directly to protein kinase Cζ (PKCζ), a regulator that binds alternatively to sp1 and phosphorylates it (13Pal S. Claffey K.P. Cohen H.T. Mukhopadhyay D. J. Biol. Chem. 1998; 273: 26277-26280Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Accordingly, it has been proposed that pVHL indirectly regulates a variety of downstream mRNA targets encoding platelet-derived growth factor B (PDGF-B), glucose transporter 1 (GLUT-1), transforming growth factor α (TGF-α), and carbonic anhydrase 9 and12 (CA9 and CA12) (1Maher E.R. Kaelin Jr., W.G. Medicine (Baltimore). 1997; 76: 381-391Crossref PubMed Scopus (434) Google Scholar, 14Ivanov S.V. Kuzmin I. Wei M.H. Pack S. Geil L. Johnson B.E. Stanbridge E.J. Lerman M.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12596-12601Crossref PubMed Scopus (341) Google Scholar, 15Pioli P.A. Rigby W.F. J. Biol. Chem. 2001; 276: 40346-40352Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Additional studies have shown that pVHL has a specific role in targeting different proteins for degradation. This role is carried out via the ubiquitylation pathway, where pVHL is associated with an active E3 ubiquitin ligase multiprotein complex (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar, 6Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (590) Google Scholar, 16Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (430) Google Scholar, 17Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4179) Google Scholar, 18Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar, 19Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1269) Google Scholar, 20Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (554) Google Scholar, 21Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar, 22Kamura T. Brower C.S. Conaway R.C. Conaway J.W. J. Biol. Chem. 2002; 277: 30388-30393Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In the context of the VCB ternary complex, the pVHL protein binds to Cul2 and Rbx1 via Elongin B and forms the VCB-CR complex, an analogue of the SCF (Skp1-Cul1-F-box) complex (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar, 8Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin Jr., W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (576) Google Scholar, 11Duan D.R. Pause A. Burgess W.H. Aso T. Chen D.Y. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (513) Google Scholar, 23Kishida T. Stackhouse T.M. Chen F. Lerman M.I. Zbar B. Cancer Res. 1995; 55: 4544-4548PubMed Google Scholar). Protein kinase Cλ and the heteronuclear ribonucleoprotein A2 (hnRNP A2) have been shown to be substrates of the VCB-CR ubiquitin ligase complex (15Pioli P.A. Rigby W.F. J. Biol. Chem. 2001; 276: 40346-40352Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Okuda H. Saitoh K. Hirai S. Iwai K. Takaki Y. Baba M. Minato N. Ohno S. Shuin T. J. Biol. Chem. 2001; 276: 43611-43617Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Moreover, it has been shown that pVHL plays a central role in the cellular response to changes in oxygen availability, as hypoxia-inducible transcription factors HIF-1α and HIF-2α have been shown to be targets of the VCB-CR ubiquitin ligase complex (6Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (590) Google Scholar, 16Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (430) Google Scholar, 17Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4179) Google Scholar, 18Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar, 19Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1269) Google Scholar, 20Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (554) Google Scholar, 21Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar, 22Kamura T. Brower C.S. Conaway R.C. Conaway J.W. J. Biol. Chem. 2002; 277: 30388-30393Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A recent study revealed a novel ubiquitylation target, the VHL-interacting deubiquitinating enzyme 1 (VDU1), which is recruited into the VCB complex and binds directly to pVHL. VDU1 is suggested to act as a protease responsible for deubiquitylation of downstream ubiquitinated targets of the VCB-CR complex (25Li Z. Na X. Wang D. Schoen S.R. Messing E.M. Wu G. J. Biol. Chem. 2002; 277: 4656-4662Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Other studies have suggested that pVHL is involved in the extracellular matrix metabolism where it directly interacts with fibronectin, and its inactivation leads to impaired extracellular fibronectin organization in VHL(–) cells (26Ohh M. Yauch R.L. Lonergan K.M. Whaley J.M. Stemmer-Rachamimov A.O. Louis D.N. Gavin B.J. Kley N. Kaelin Jr., W.G. Iliopoulos O. Mol. Cell. 1998; 1: 959-968Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Another recent study has proposed that pVHL regulates integrins and is essential for the formation of β1 fibrillar adhesions, which are involved in cytoskeletal rearrangement and required for the organization of fibronectin (27Esteban-Barragan M.A. Avila P. Alvarez-Tejado M. Gutierrez M.D. Garcia-Pardo A. Sanchez-Madrid F. Landazuri M.O. Cancer Res. 2002; 62: 2929-2936PubMed Google Scholar). Additionally, the pVHL protein has been associated with regulation of the stability of microtubules and their protection from depolymerization through direct interaction (28Hergovich A. Lisztwan J. Barry R. Ballschmieter P. Krek W. Nat. Cell Biol. 2003; 5: 64-70Crossref PubMed Scopus (289) Google Scholar). Another stabilization role was observed with the short-lived protein Jade-1 (gene for apoptosis and differentiation in epithelia). This protein, which is highly expressed in the kidney, is stabilized by its strong interaction with the pVHL protein (29Zhou M.I. Wang H. Ross J.J. Kuzmin I. Xu C. Cohen H.T. J. Biol. Chem. 2002; 277: 39887-39898Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Finally, the unassembled form of pVHL is associated with two different types of molecular chaperones, Hsp70 and TriC/CCT (30Feldman D.E. Thulasiraman V. Ferreyra R.G. Frydman J. Mol. Cell. 1999; 4: 1051-1061Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 31Melville M.W. McClellan A.J. Meyer A.S. Darveau A. Frydman J. Mol. Cell. Biol. 2003; 23: 3141-3151Crossref PubMed Scopus (110) Google Scholar). The biological significance of pVHL is evident by its evolutionary conservation. Homologues of the mammalian pVHL protein are found in low invertebrates such as Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans. As more complete eukaryotic genomes are sequenced, we will expect to find additional pVHL homologues along the evolutionary tree. The understanding of pVHL structure-function relationship in the context of its unbound solution structure is therefore highly important. Such studies are significant in the wider context of structure and stability of other tumor suppressor proteins as well. The wild-type p53 tumor suppressor protein was found to be relatively unstable; its isolated core domain had moderate thermodynamic stability with relatively small changes leading to loss of function (32Bullock A.N. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14338-14342Crossref PubMed Scopus (356) Google Scholar). Likewise, the wild-type p16(INK4a) tumor suppressor protein was found to have low stability and to be highly vulnerable to mutations, which altered its secondary structure and global fold (33Tevelev A. Byeon I.J. Selby T. Ericson K. Kim H.J. Kraynov V. Tsai M.D. Biochemistry. 1996; 35: 9475-9487Crossref PubMed Scopus (76) Google Scholar, 34Tang K.S. Guralnick B.J. Wang W.K. Fersht A.R. Itzhaki L.S. J. Mol. Biol. 1999; 285: 1869-1886Crossref PubMed Scopus (114) Google Scholar). These findings may imply that intrinsic instability could be a functional trait in tumor suppressors and may be the means by which these key role proteins modulate their activity. Here, we examine the secondary structure, hydrodynamic characteristics, core packing, and thermodynamic stability of the pVHL tumor suppressor protein in solution under physiologically relevant conditions by using biophysical techniques. The results of our studies, as presented here, allow better understanding of the role of the pVHL protein in various cellular processes and may shed light on the general paradigm of tumor suppressor proteins. Protein Expression and Purification—The BL21(DE3) strain of Escherichia coli was co-transformed by electroporation (Bio-Rad Micro-Pulser) with two plasmids that were kindly provided to us by Dr. Nikola Pavletich (Memorial Sloan-Kettering Cancer Center, New York). The first plasmid, pGEX-4T-3, includes the human VHL gene (residues 54 to 213) fused to glutathione S-transferase (GST) and the full-length human Elongin B gene as a dicistronic massage. The second plasmid, pBB75, includes the human Elongin C gene (residues 17 to 112). Transformed cells were grown at 37 °C, 200 rpm, in 2XYT medium under dual antibiotic selection (100 μg/ml ampicillin and 30 μg/ml kanamycin). Cells were induced overnight at 25 °C with two doses of 1 mm isopropyl-1-thio-β-d-galactopyranoside and supplementary ampicillin (100 μg/ml); one dose was applied at A600 = 0.8–1, and a second dose was applied after 6 h of induction. Cells were harvested by centrifugation, and the pellets were frozen at –70 °C before purification. Cell pellets were resuspended in suspension buffer (50 mm Tris-HCl, pH 8, 200 mm NaCl, 10 mm dithiothreitol, 5 mm phenylmethylsulfonyl fluoride, and mixture protease inhibitor), and suspended cells were lysed by a French press cell disruptor at 14,000 p.s.i. The lysate was treated with DNase (10 units/ml) and centrifuged for 30 min, at 4 °C. Supernatant was filtered through a 0.45 μm filter (Corning) and subjected to GSTrap FF 1-ml column (Amersham Biosciences) using the ÄKTA prime automated liquid chromatography system (Amersham Biosciences). The GST-pVHL fusion protein was treated on column with thrombin protease (Amersham Biosciences) overnight to cleave the GST moiety, and purified pVHL was eluted with 50 mm Tris-HCl, pH 8, and 200 mm NaCl. Purification was assessed by Coomassie staining of SDS/PAGE. Protein concentration was determined from absorbance at 280 nm using ϵ280 nm = 17,900 cm–1m–1. CD Spectra Measurements—CD spectra at far- (200–250 nm) and near-UV (250–320 nm) were recorded with an AVIV 202 spectropolarimeter (Aviv Instruments, Lakewood NJ) equipped with a temperature-controlled cell using a cell of pathlength 0.5 cm; bandwidth was 1 nm, and averaging time was 30 s for each measurement. Protein concentration was 3 μm (far-UV) and 7 μm (near-UV) in a buffer containing 50 mm Tris-HCl (pH 8) and 200 mm NaCl. Chemical Denaturation—Urea denaturation studies were carried out using CD. A solution of 3 μm protein in buffer (50 mm Tris-HCl, pH 8, and 200 mm NaCl) was mixed stepwise with appropriate amounts of the same solution containing 6 m urea to achieve the appropriate concentration of protein and denaturant for the chemical denaturation. Changes in ellipticity were scanned from 222 to 218 nm for each step of urea addition; bandwidth was 1 nm, averaging time was 30 s, and cell pathlength was 0.5 cm. Measurements were conducted at 37 and 25 °C. Size Exclusion Chromatography—Gel filtration experiments were performed using a Sephacryl S100 16/60 column (Amersham Pharmacia Biotech) with a separation range of 1–100 kDa connected to an ΔKTA prime automated liquid chromatography system (Amersham Biosciences). The running buffer used was 50 mm Tris-HCl pH 8, and 200 mm NaCl. The column was calibrated using gel filtration low molecular weight standards (Amersham Biosciences). A 1-ml sample at a final protein concentration of 8 μm was chromatographically analyzed using a flow rate of 0.5 ml/min. Absorbance was monitored at 280 nm, and elution volumes were determined from UV chromatogram. The partition coefficient, Kav, was calculated from the elution volume of the sample, Ve, and total bed volume, Vt, using the expression: Kav = (Ve – V0)/(Vt – V0). Calibration curves and equations were established. Dynamic Light-scattering Measurements—Hydrodynamic radius (RH) measurements were made at 25 °C with a DynaPro MSTC800 instrument (Protein Solutions Inc., Charlottesville, VA). A sample (50 μl) containing 0.3 mg/ml protein in buffer (50 mm Tris-HCl, pH 8, and 200 mm NaCl) was centrifuged and filtered to avoid dust particles. The sample was placed directly in a quartz cuvette, and the light-scattering intensity was collected at an angle of 90° using a 10-s acquisition time at 74% laser power. Particle diffusion coefficient was calculated from auto-correlated light intensity data and converted to RH with the Stokes-Einstein equation. A histogram of the percentage of the scattering mass versus RH was calculated using Dynamics data analysis software (Protein Solutions). 8-Anilino-1-naphthalene Sulfonic Acid (ANSA) Fluorescence Studies—Fluorescence emission spectra of two solutions were generated with excitation wavelength of 350 nm. The first solution contained 3 μm ANSA in buffer (50 mm Tris-HCl, pH 8, and 200 mm NaCl). The second solution was similar and contained protein at a final concentration of 3 μm. Samples were equilibrated at room temperature. The fluorescence emission spectra were recorded (Luminescence Spectrometer LS50B, PerkinElmer Life Sciences). The cuvette length was 1 cm. Measurements were conducted at room temperature. Baseline corrections were made with buffer lacking protein and ANSA, but otherwise identical. Analytical Ultracentrifugation—Sedimentation equilibrium experiments were performed overnight at 20 °C on a Beckman Optima XL-A analytical ultracentrifuge (Beckman Instruments) equipped with 12-mm Epon double sector cells in an An-60 Ti rotor. Protein was analyzed in PBS buffer (pH 7.4). Sedimentation equilibrium scans were carried out at 21,000 rpm. Absorbance of sedimenting material was assayed at 232 nm. Molecular masses were evaluated from ln A versus r2 plots employing the SEGAL computer program based on the concept of numerical fitting of the sedimentation equilibrium pattern where A is the absorbance and r is the distance from the rotor center. A partial specific volume of 0.73 cm3/g was used for all calculations. Fluorescence Labeling and Spectroscopy—Purified VHL protein was reacted with two equivalents of N-hydroxysuccinimide fluorescein ester (Pierce) in PBS (pH 8) for 24 h at room temperature in the dark as described (35Gazit E. Burshtein N. Ellar D.J. Sawyer T. Shai Y. Biochemistry. 1997; 36: 15546-15554Crossref PubMed Scopus (32) Google Scholar). Unreacted dye was separated from labeled protein by gel filtration chromatography on a MicroSpin G-50 Sephadex column (Amersham Biosciences). Fluorescence emission and anisotropy were monitored in PBS using a PerkinElmer Life Sciences LS50B fluorescence spectrophotometer. Although the crystal structure of pVHL was determined in the context of the ternary complex with Elongin proteins using x-ray crystallography (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar, 6Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (590) Google Scholar), the structure of the protein in solution and its unbound structure have not yet been studied. To gain insight on the structure of the protein under such conditions, we studied the secondary and tertiary structure, the hydrodynamic properties, the thermal and chemical stability, and the core packing of pVHL under near-physiological solvent conditions using biophysical techniques. Determination of Secondary and Tertiary Structure by Circular Dichroism—Initial information regarding the secondary and tertiary structure of the purified, unbound pVHL in solution was obtained by CD. The far-UV CD spectra of pVHL at 25 °C, shown in Fig. 1A, reveals spectral signals consistent with a protein comprising an intact secondary structure, rendering both α-helices and β-sheets. The α-helical and β-sheet content estimated by deconvolution of the CD spectra (k2d; www.embl-heidelberg.de/~andrade/k2d) shows 29% α-helix content and 34% β-sheet content. This is consistent with the secondary structure of the bound pVHL protein as determined previously by x-ray crystallography (5Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar). The near-UV CD spectra of pVHL at 25 °C shown in Fig. 1B reveals no significant signals originating from aromatic side chains, thus suggesting a dramatic loss of tertiary structure of the soluble and unbound protein in comparison with the complex-related crystal structure. Chemical Denaturation—The thermodynamic instability of tumor suppressor proteins appears to be a common structural denominator (32Bullock A.N. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14338-14342Crossref PubMed Scopus (356) Google Scholar, 33Tevelev A. Byeon I.J. Selby T. Ericson K. Kim H.J. Kraynov V. Tsai M.D. Biochemistry. 1996; 35: 9475-9487Crossref PubMed Scopus (76) Google Scholar, 34Tang K.S. Guralnick B.J. Wang W.K. Fersht A.R. Itzhaki L.S. J. Mol. Biol. 1999; 285: 1869-1886Crossref PubMed Scopus (114) Google Scholar). We therefore studied the thermodynamic stability of purified pVHL using urea melt at two different temperatures (37 and 25 °C). The degree of denaturation was assessed by monitoring ellipticity changes at 222 and 218 nm as a function of urea concentrations. As can be observed in Fig. 2, the unfolding of pVHL occurs at fairly low urea concentrations. Unfolding as reflected by a significant reduction in ellipticity was observed at urea concentrations as low as 0.25 m at both temperatures. Furthermore, the unfolding event was partially irreversible, and the formation of amorphous aggregates could be visualized by the naked eye. Therefore, no exact thermodynamic parameters could be calculated. However, the linear nature of the initial slope of the curve and the aggregative behavior implies that unbound pVHL stability is marginal, as was observed previously with other tumor suppressor proteins (33Tevelev A. Byeon I.J. Selby T. Ericson K. Kim H.J. Kraynov V. Tsai M.D. Biochemistry. 1996; 35: 9475-9487Crossref PubMed Scopus (76) Google Scholar). Size Exclusion Chromatography—The marginal stability of pVHL, as observed in the chemical denaturation experiments, raised the possibility that pVHL might be partially unfolded in its unbound state. Size exclusion chromatography was therefore used to estimate the hydrodynamic dimensions of the protein and as a probe for elucidating the compactness of the protein's tertiary structure, because the elution volume of partially or fully unfolded proteins is significantly smaller than that of well folded proteins due to the large increase in the Stokes radius. We performed size exclusion chromatography in 50 mm Tris-HCl, pH 8, containing 200 mm NaCl. The pVHL protein was eluted at a volume of 58.5 ml on a calibrated column (as described under “Experimental Procedures”), which is very close to the ovalbumin standard (Mw = 43,000 Da) el" @default.
• W2079347968 created "2016-06-24" @default.
• W2079347968 creator A5013588863 @default.
• W2079347968 creator A5082716768 @default.
• W2079347968 date "2004-04-01" @default.
• W2079347968 modified "2023-10-16" @default.
• W2079347968 title "The von Hippel-Lindau Tumor Suppressor Protein Is a Molten Globule under Native Conditions" @default.
• W2079347968 cites W1512176015 @default.
• W2079347968 cites W1720678970 @default.
• W2079347968 cites W1749022239 @default.
• W2079347968 cites W1970078605 @default.
• W2079347968 cites W1972009030 @default.
• W2079347968 cites W1972032694 @default.
• W2079347968 cites W1973341288 @default.
• W2079347968 cites W1981124017 @default.
• W2079347968 cites W1985179172 @default.
• W2079347968 cites W1987825597 @default.
• W2079347968 cites W1988935612 @default.
• W2079347968 cites W1990818598 @default.
• W2079347968 cites W1991930213 @default.
• W2079347968 cites W1996722842 @default.
• W2079347968 cites W1998735636 @default.
• W2079347968 cites W2000789117 @default.
• W2079347968 cites W2012398004 @default.
• W2079347968 cites W2014945371 @default.
• W2079347968 cites W2016579573 @default.
• W2079347968 cites W2018304885 @default.
• W2079347968 cites W2023952710 @default.
• W2079347968 cites W2024954553 @default.
• W2079347968 cites W2031485253 @default.
• W2079347968 cites W2031514188 @default.
• W2079347968 cites W2032677592 @default.
• W2079347968 cites W2043993990 @default.
• W2079347968 cites W2054177557 @default.
• W2079347968 cites W2059493348 @default.
• W2079347968 cites W2080374319 @default.
• W2079347968 cites W2081288740 @default.
• W2079347968 cites W2084679948 @default.
• W2079347968 cites W2087841059 @default.
• W2079347968 cites W2089289562 @default.
• W2079347968 cites W2090754983 @default.
• W2079347968 cites W2093622484 @default.
• W2079347968 cites W2093776019 @default.
• W2079347968 cites W2109034041 @default.
• W2079347968 cites W2119693327 @default.
• W2079347968 cites W2120881407 @default.
• W2079347968 cites W2125505202 @default.
• W2079347968 cites W2138586777 @default.
• W2079347968 cites W2145538573 @default.
• W2079347968 cites W2147718842 @default.
• W2079347968 cites W2149268957 @default.
• W2079347968 cites W2172170481 @default.
• W2079347968 cites W2596045242 @default.
• W2079347968 cites W3022518945 @default.
• W2079347968 cites W4210968583 @default.
• W2079347968 doi "https://doi.org/10.1074/jbc.m311225200" @default.
• W2079347968 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14963040" @default.
• W2079347968 hasPublicationYear "2004" @default.
• W2079347968 type Work @default.
• W2079347968 sameAs 2079347968 @default.
• W2079347968 citedByCount "35" @default.
• W2079347968 countsByYear W20793479682012 @default.
• W2079347968 countsByYear W20793479682013 @default.
• W2079347968 countsByYear W20793479682014 @default.
• W2079347968 countsByYear W20793479682015 @default.
• W2079347968 countsByYear W20793479682016 @default.
• W2079347968 countsByYear W20793479682017 @default.
• W2079347968 countsByYear W20793479682018 @default.
• W2079347968 countsByYear W20793479682019 @default.
• W2079347968 countsByYear W20793479682020 @default.
• W2079347968 countsByYear W20793479682021 @default.
• W2079347968 countsByYear W20793479682022 @default.
• W2079347968 countsByYear W20793479682023 @default.
• W2079347968 crossrefType "journal-article" @default.
• W2079347968 hasAuthorship W2079347968A5013588863 @default.
• W2079347968 hasAuthorship W2079347968A5082716768 @default.
• W2079347968 hasBestOaLocation W20793479681 @default.
• W2079347968 hasConcept C104317684 @default.
• W2079347968 hasConcept C118493188 @default.
• W2079347968 hasConcept C12554922 @default.
• W2079347968 hasConcept C179185449 @default.
• W2079347968 hasConcept C185592680 @default.
• W2079347968 hasConcept C47701112 @default.
• W2079347968 hasConcept C55493867 @default.
• W2079347968 hasConcept C86803240 @default.
• W2079347968 hasConceptScore W2079347968C104317684 @default.
• W2079347968 hasConceptScore W2079347968C118493188 @default.
• W2079347968 hasConceptScore W2079347968C12554922 @default.
• W2079347968 hasConceptScore W2079347968C179185449 @default.
• W2079347968 hasConceptScore W2079347968C185592680 @default.
• W2079347968 hasConceptScore W2079347968C47701112 @default.
• W2079347968 hasConceptScore W2079347968C55493867 @default.
• W2079347968 hasConceptScore W2079347968C86803240 @default.
• W2079347968 hasIssue "17" @default.
• W2079347968 hasLocation W20793479681 @default.
• W2079347968 hasOpenAccess W2079347968 @default.
• W2079347968 hasPrimaryLocation W20793479681 @default.
• W2079347968 hasRelatedWork W1531601525 @default.

• next