HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.
HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.
Krell
Tino
T
Aventis Pasteur, 1541 avenue Marcel Meriueux, 69280 Marcy l'Etoile, France. tino.krell@aventis.com
Greco
Frédéric
F
Engel
Olivier
O
Dubayle
Jean
J
Dubayle
Joseline
J
Kennel
Audrey
A
Charloteaux
Benoit
B
Brasseur
Robert
R
Chevalier
Michel
M
Sodoyer
Regis
R
El Habib
Raphaëlle
R
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004355:Drug Stability; D006031:Glycosylation; D019302:HIV Envelope Protein gp160 / Q000737:chemistry* / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013816:Thermodynamics
D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins