Expression, purification, and activities of full‐length and truncated versions of the integral membrane protein Vpu from HIV‐1

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.
Protein Science (Impact Factor: 2.86). 04/2002; 11(3):546-57. DOI: 10.1110/ps.37302
Source: PubMed

ABSTRACT Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.

Download full-text


Available from: Stephan Bour, Jul 28, 2015
  • Source
    • "In the Mycobacterium tuberculosis genome 60% of the putative membrane proteins have a molecular weight less than 40 kDa [1]. Furthermore, there is growing evidence that the membrane and water-soluble domains of membrane proteins often function independently [2] [3] [4] [5] [6] [7]. Consequently, there are many hydrophobic peptides and proteins that are important to express in high yields for structural and functional studies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione-S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg2+ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies.
    Biochimica et Biophysica Acta 05/2008; 1778(4):1060-6. DOI:10.1016/j.bbamem.2007.12.024 · 4.66 Impact Factor
  • Source
    • "Indeed, throughout the spectra the overlap clearly shows a uniformity of structure and environment for these three constructs. In the structural study of Vpu by solution NMR, the same phenomenon was observed when the spectra of the transmembrane peptide and that of the full-length protein were overlaid (Marassi et al. 1999; Ma et al. 2002). For CorA–TM2, the spectral overlap suggests that the interaction between TM1 and TM2 is relatively weak so that the helical backbone structures are essentially independent of each other. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.
    Protein Science 11/2007; 16(10):2153-65. DOI:10.1110/ps.072996707 · 2.86 Impact Factor
  • Source
    • "As the aim of this study was to use the most efficient and high-throughput methods for evaluating production of integral membrane proteins, we chose to evaluate protein production levels based on analysis of whole cell lysates, as it is clear from the above evidence that the membrane transporters in this study, most of which have at least eight predicted transmembrane helices, and only three of which have three predicted helices, will most likely be partially or fully localized to the membrane. In addition, while production in the membrane is preferred, there are several studies in which in vitro refolding of eukaryotic integral membrane proteins has been successful (Kiefer et al. 1996; Rogl et al. 1998; Ma et al. 2002; for review, see Kiefer 2003), and thus it may be possible to purify protein from inclusion bodies as well. An outline of the expression strategy is shown in Figure 1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.
    Protein Science 10/2006; 15(9):2178-89. DOI:10.1110/ps.062312706 · 2.86 Impact Factor
Show more