Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant

The denatured state of HIV-1 protease under native conditions

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high‐resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV‐1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant‐free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV‐1 protease under native conditions displays rich patterns of transient native and non‐native structures, which could be of relevance to its guidance through a complex folding process

GAGE cancer-germline antigens are recruited to the nuclear envelope by germ cell-less (GCL)

GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209-320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells

The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human α-lactalbumin (α-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. α-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the α-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the α-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the α-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the α-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of α-LA. The pH-dependent effects reported here for α-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins

GAGE proteins bind dsDNA.

<p>A–B. Western blot of pulldown experiments testing the binding of recombinant GAGE12I (A) or endogenous GAGE proteins from MZ2-MEL lysates (B) to cellulose alone, or cellulose-immobilized native calf thymus dsDNA. C. Electrophoretic mobility shift assays. Each indicated concentration of purified GAGE12I was incubated with the ³²P-labelled 90-bp EcoRI-PvuII DNA fragment from pUC19 at 1 pg/µl; samples were then resolved by agarose gel electrophoresis. D. Same experiment as in (C), but with addition of NaCl. E. Prediction of putative DNA binding amino acids in GAGE12I using the program BindN. +/− = binding/no binding; 0–9 = confidence of binding prediction.</p

GAGE proteins interact with GCL.

<p>A. GAGE12I was used as bait in a yeast two-hybrid screen of a testis cDNA library. Plasmids encoding putative preys identified in the primary screen were rescued and retransformed into EGY42 with the bait GAGE12I vector (B) or control vector (C). Clone D2 exhibited growth on Ura- medium and blue coloration on X-Gal medium, indicating an interaction between bait and prey. The encoded prey polypeptide of D2 comprised human GCL residues 84–320, which includes the proposed BTB/POZ and BACK domains. B. Interaction between GCL and GAGE12I was verified by Lumier pull-downs from HEK293 cells co-transfected with Protein A-fused GAGE and Luciferase-fused GCL. Normalized luciferase signals (bound/input) are presented as Z scores, representing the number of standard deviations from the mean of a large set of independently derived, non-interacting Lumier protein pairs (see Materials and Methods for details). C. Pull-down assay with recombinant His-tagged GAGE12I and GST-GCL.</p

GCL and GAGE proteins are co-expressed in human cancer cells lines.

<p>GCL and GAGE protein expression was examined in 9 human cancer cells lines derived from cervix (HeLa), colon (HCT116), melanoma (MZ2-MEL), breast (BrCa-MZ01, SK-BR3, T47D, M4A4, MCF7) and embryonic cancer (NTERA2) using Western blotting. A375 melanoma cells with exogenous expression of GCL were included as positive control. Antibodies: GCL pAb1, Sigma Aldrich; GCL pAb2, clone A14, Santa Cruz Biotech; GAGE mAb, clone M3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045819#pone.0045819-Gjerstorff2" target="_blank">[4]</a>, beta-actin mAb, Ab6276; Abcam.</p