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1.
Bioinformatics ; 34(12): 2053-2060, 2018 06 15.
Article in English | MEDLINE | ID: mdl-29365182

ABSTRACT

Motivation: Polypeptide sequence length is the single dominant factor hampering the effectiveness of currently available software tools for de novo calculation of amino acid-specific protonation constants in disordered polypeptides. Results: We have developed pepKalc, a robust simulation software for the comprehensive evaluation of protein electrostatics in unfolded states. Our software completely removes the limitations of the previously reported Monte-Carlo approaches in the computation of protein electrostatics by using a hybrid approach that effectively combines exact and mean-field calculations to rapidly obtain accurate results. Paired with a modern architecture GPU, pepKalc is capable of evaluating protonation behavior for an arbitrary-size polypeptide in a sub-second time regime. Availability and implementation: http://protein-nmr.org and https://github.com/PeptoneInc/pepkalc.


Subject(s)
Computational Biology/methods , Peptides/chemistry , Software , Static Electricity , Humans , Monte Carlo Method , Peptides/metabolism , alpha-Synuclein/chemistry
2.
Biophys J ; 112(10): 2109-2116, 2017 May 23.
Article in English | MEDLINE | ID: mdl-28538148

ABSTRACT

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pKa determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow.


Subject(s)
Bacterial Proteins/metabolism , Photoreceptors, Microbial/metabolism , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Bacterial Proteins/chemistry , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Halorhodospira halophila , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Photoreceptors, Microbial/chemistry , Protons
3.
J Biomol NMR ; 62(2): 129-42, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25820763

ABSTRACT

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T 1 relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T 1 values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α1-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Peptides/chemistry , alpha-Synuclein/chemistry , Ferric Compounds/chemistry , Humans , Models, Theoretical , Protein Conformation , Protons , Solutions
4.
Biophys J ; 102(3): 579-86, 2012 Feb 08.
Article in English | MEDLINE | ID: mdl-22325281

ABSTRACT

Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated (13)C(γ) resonances in sensitive and well-resolved two-dimensional (13)C-(1)H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK(a) values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41-11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.


Subject(s)
Bacterial Proteins/chemistry , Chemical Phenomena , Nuclear Magnetic Resonance, Biomolecular , Photoreceptors, Microbial/chemistry , Tyrosine , Bacterial Proteins/metabolism , Crystallography, X-Ray , Halorhodospira halophila , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Photoreceptors, Microbial/metabolism , Protein Structure, Secondary , Protons , Static Electricity
5.
Biomol NMR Assign ; 6(1): 15-8, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21647611

ABSTRACT

Neuroligins act as heterophilic adhesion molecules at neuronal synapses. Their cytoplasmic domains interact with synaptic scaffolding proteins, and have been shown to be intrinsically disordered. Here we report the backbone and side chain (1)H, (13)C and (15)N resonance assignments for the cytoplasmic domain of human neuroligin 3.


Subject(s)
Cell Adhesion Molecules, Neuronal/chemistry , Cytoplasm/metabolism , Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Cell Adhesion Molecules, Neuronal/metabolism , Humans , Membrane Proteins/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Protein Structure, Tertiary
6.
J Biomol NMR ; 51(1-2): 199-207, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21947928

ABSTRACT

Here we describe phasing anomalies observed in gradient sensitivity enhanced (15)N-(1)H HSQC spectra, and analyze their origin. It is shown that, as a result of (15)N off-resonance effects, dispersive contributions to the (1)H signal become detectable, and lead to (15)N-offset dependent phase errors. Strategies that effectively suppress these artifacts are presented.


Subject(s)
Artifacts , Magnetic Resonance Spectroscopy/methods , Reproducibility of Results
7.
Biomol NMR Assign ; 5(1): 79-84, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21069485

ABSTRACT

Here we present the 100% complete assignment chemical shift of non-labile (1)H, (15)N and (13)C nuclei of Calbindin D(9k) P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D(9k) extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858-14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464-6465, 1994; Yamazaki et al. in Biochem 32:5656-5669, 1993b).


Subject(s)
Amino Acid Substitution , Calcium/metabolism , S100 Calcium Binding Protein G/chemistry , Amino Acid Sequence , Animals , Calbindins , Carbon Isotopes , Cattle , Hydrogen , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular
8.
J Mol Biol ; 353(4): 859-72, 2005 Nov 04.
Article in English | MEDLINE | ID: mdl-16213522

ABSTRACT

The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121 residue excreted virulence factor. It acts by binding the C5a- (C5aR) and formylated peptide receptor (FPR) and thereby blocks specific phagocyte responses. Here, we report the solution structure of a CHIPS fragment consisting of residues 31-121 (CHIPS31-121). CHIPS31-121 has the same activity in blocking the C5aR compared to full-length CHIPS, but completely lacks FPR antagonism. CHIPS31-121 has a compact fold comprising an alpha-helix (residues 38-51) packed onto a four-stranded anti-parallel beta-sheet. Strands beta2 and beta3 are joined by a long loop with a relatively well-defined conformation. Comparison of CHIPS31-121 with known structures reveals striking homology with the C-terminal domain of staphylococcal superantigen-like proteins (SSLs) 5 and 7, and the staphyloccocal and streptococcal superantigens TSST-1 and SPE-C. Also, the recently reported structures of several domains of the staphylococcal extracellullar adherence protein (EAP) show a high degree of structural similarity with CHIPS. Most of the conserved residues in CHIPS and its structural homologues are present in the alpha-helix. A conserved arginine residue (R46 in CHIPS) appears to be involved in preservation of the structure. Site-directed mutagenesis of all positively charged residues in CHIPS31-121 reveals a major involvement of arginine 44 and lysine 95 in C5aR antagonism. The structure of CHIPS31-121 will be vital in the further unraveling of its precise mechanism of action. Its structural homology to S.aureus SSLs, superantigens, and EAP might help the design of future experiments towards an understanding of the relationship between structure and function of these proteins.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Enterotoxins , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Receptors, Formyl Peptide , Staphylococcus aureus/chemistry , Superantigens , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Enterotoxins/chemistry , Enterotoxins/immunology , Enterotoxins/metabolism , Iodine Radioisotopes/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phagocytosis , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Formyl Peptide/chemistry , Receptors, Formyl Peptide/immunology , Receptors, Formyl Peptide/metabolism , Sequence Homology, Amino Acid , Superantigens/chemistry , Superantigens/immunology , Superantigens/metabolism
9.
Biophys J ; 89(1): 201-10, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15879478

ABSTRACT

Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with K(D)-values in the nano- and micromolar range, respectively. However, reported K(D)-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in K(D)-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol.


Subject(s)
Escherichia coli/enzymology , Mannitol/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Absorption , Binding Sites , Biological Transport , Computer Simulation , Detergents/pharmacology , Dose-Response Relationship, Drug , Escherichia coli Proteins , Genetic Complementation Test , Kinetics , L-Lactate Dehydrogenase/chemistry , Ligands , Mannitol/chemistry , Membrane Transport Proteins/chemistry , Monosaccharide Transport Proteins , Phosphoenolpyruvate/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphorylation , Plasmids/metabolism , Polyethylene Glycols , Protein Binding , Proteins/chemistry , Thrombin/chemistry , Time Factors , Ultraviolet Rays
10.
J Struct Biol ; 146(3): 272-80, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15099569

ABSTRACT

The backbone dynamic properties of uniformly (15)N-labeled calcium-saturated calmodulin (Ca(2+)-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by (15)N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca(2+)-CaM, which is a long, solvent-exposed alpha-helix in the crystal structure but is known to be partially unwound and flexible in solution. (15)N T(1), T(2), and (15)N-[(1)H] NOE values were determined for both Ca(2+)-CaM in H(2)O and Ca(2+)-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78-Glu80 of 0.29, 0.17, and 0.27 in H(2)O and 0.82, 0.66, and 0.64 in 35% TFE. However, (15)N-[(1)H] NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78-Glu80 (15)N-[(1)H] NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca(2+)-CaM in 35% TFE, with motional correlation times of approximately 10 and approximately 9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca(2+)-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.


Subject(s)
Calcium/chemistry , Calmodulin/chemistry , Nuclear Magnetic Resonance, Biomolecular , Kinetics , Motion , Nitrogen Isotopes , Protein Conformation/drug effects , Protein Structure, Secondary/drug effects , Recombinant Proteins , Trifluoroethanol/pharmacology , Water
11.
Biophys J ; 86(4): 1959-68, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15041640

ABSTRACT

Flow dialysis has found widespread use in determining the dissociation constant (KD) of a protein-ligand interaction or the amount of available binding sites (E0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the KD and E0 is presented, together with an extensive error analysis of the method. The flow-dialysis technique is experimentally simple to perform. However, a number of practical aspects of this method can have a large impact on the outcome of KD and E0. We have investigated all sources of significant systematic and random errors, using the interaction between mannitol and its transporter from Escherichia coli as a model. Monte Carlo simulations were found to be an excellent tool to assess the impact of these errors on the binding parameters and to define the experimental conditions that allow their most accurate estimation.


Subject(s)
Computer Simulation , Ligands , Microdialysis/methods , Monte Carlo Method , Proteins/chemistry , Protein Binding , Ultrafiltration
13.
J Mol Biol ; 330(2): 397-407, 2003 Jul 04.
Article in English | MEDLINE | ID: mdl-12823977

ABSTRACT

We report the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, HPr, in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue. The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues, as (i) the observation of transient histidine photo-CIDNP signals during refolding of three of the mutants (F2W, F29W, and F48W) indicates a strong decrease in tryptophan accessibility to the flavin dye; (ii) iodide quenching experiments show that the quenching of the fluorescence of F48W is less efficient for the species formed during the dead-time of the stopped-flow experiment than for the fully native state; and (iii) kinetic fluorescence anisotropy measurements show that the tryptophan side-chain of F48W has lower mobility in the dead-time intermediate state than in both the fully denatured and fully native states. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues. This process may be important in preventing the protein from aggregating prior to the acquisition of native-like structure in which hydrophobic residues are exposed in order to play their role in the function of the protein. The phenylalanine residue at position 48 is likely to be of particular interest in this regard as it is involved in the binding to enzymes I and II that mediates the transfer of a phosphoryl group between the two enzymes.


Subject(s)
Bacterial Proteins , Fluorescence Polarization/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Photochemistry , Protein Denaturation , Protein Folding , Tryptophan/chemistry
14.
Biochemistry ; 42(17): 4883-95, 2003 May 06.
Article in English | MEDLINE | ID: mdl-12718529

ABSTRACT

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.


Subject(s)
Bacterial Proteins , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Tryptophan , Amino Acid Substitution , Binding Sites , Fluorescent Dyes , Guanidine , Histidine , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Thermodynamics
15.
J Biomol NMR ; 23(3): 169-79, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12238589

ABSTRACT

Residual dipolar couplings between 15N and 1H nuclear spins in HPr were used to determine the protein's orientation in a medium of bicelles, oriented by a magnetic field. In the case of wild-type HPr the protein's non-spherical shape can explain its orientation in this medium. In the case of the F48W mutant it was found that at least one other mechanism contributes to the observed orientation of the protein, to a degree that depends on the concentration of phosphate ions in the medium. We propose that the F48W mutant has a weak affinity towards the bicelle-surfaces that decreases with increasing phosphate concentrations. We used an order-parameter description to analyse this situation and to determine the axis of main order and the sign of the order parameter pertaining to this additional orientation mechanism.


Subject(s)
Lipid Bilayers/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Anisotropy , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Lipid Bilayers/metabolism , Magnetics , Models, Chemical , Models, Molecular , Mutation, Missense , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism
16.
J Biomol NMR ; 23(3): 181-94, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12238590

ABSTRACT

Three methods for calculating nuclear magnetic resonance cross-relaxation rates from molecular dynamics simulations of small flexible molecules have been compared in terms of their ability to reproduce relaxation data obtained experimentally and to produce consistent descriptions of the system. The importance of the accuracy of the simulation versus the amount of sampling of phase space has also been assessed by comparing different length simulations performed with different time step schemes. A nine-residue peptide from the protein HPr of E. coli was used as a test system. The work shows that, in this case, single conformations or a limited ensemble of configurations are insufficient to properly describe the behavior of the peptide and that different approaches to incorporate molecular motions lead to significant differences in the cross-relaxation rates calculated. The correlation between the cross-relaxation rates calculated from simulations performed with different time step schemes was high and increased with increasing simulation length indicating that the extent of sampling is more important than the details of the atomic motion.


Subject(s)
Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Bacterial Proteins/chemistry , Fourier Analysis , Magnetics , Motion , Peptide Fragments/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein Conformation
17.
Protein Expr Purif ; 25(3): 400-8, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12182819

ABSTRACT

Herpes simplex virus type 1 and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell. Availability of substantial amounts of these proteins, or large fragments thereof, will be needed to allow studies at the molecular level. We studied the potency of the Pichia pastoris yeast expression system to produce soluble forms of gD. The DNA sequences encoding the extracellular domains of gD [amino acids 1-314 (gD-1(1-314)) and amino acids 1-254 (gD-1(1-254)) of gD-1 and amino acids 1-314 of gD-2 (gD-2(1-314))] were cloned into the P. pastoris yeast expression vector pPIC9. Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1(1-314His) and gD-1(1-254His)) to facilitate their purification. Large amounts of gD-1(1-314) and gD-1(1-314His) (280-300mg/L induction medium) were produced. The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L. SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa. The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, and AP7). In addition, we showed that gD-1(1-314), gD-2(1-314), and gD-1(1-254His) were able to interfere with binding of HSV to susceptible cells. These results indicate that the conformations of the recombinant proteins closely resemble those of native gD.


Subject(s)
Herpesvirus 1, Human/genetics , Herpesvirus 2, Human/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Pichia/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genetic Vectors , Herpesvirus 1, Human/chemistry , Herpesvirus 2, Human/chemistry , Peptide Fragments/biosynthesis , Peptide Fragments/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solubility , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/isolation & purification
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