Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Burda P, Crosskey T, Lauk K, Zurborg A, Söhnchen C, Liffner B, Wilcke L, Pietsch E, Strauss J, Jeffries C, Svergun D, Wilson D, Wilmanns M, Gilberger T, Cell Reports 31(12):107817 (2020) DOI

SASDH92 – Plasmodium falciparum lipocalin (PF3D7_0925900): Dimer-tetramer equilibrium through a concentration series (combined batch and SEC-SAXS measurements)

Plasmodium falciparum Lipocalin
MWexperimental 83 kDa
MWexpected 89 kDa
VPorod 126 nm3
log I(s) 4.72×10-2 4.72×10-3 4.72×10-4 4.72×10-5
Plasmodium falciparum Lipocalin small angle scattering data  s, nm-1
ln I(s)
Plasmodium falciparum Lipocalin Guinier plot ln 4.73×10-2 Rg: 3.2 nm 0 (3.2 nm)-2 s2
(sRg)2I(s)/I(0)
Plasmodium falciparum Lipocalin Kratky plot 1.104 0 3 sRg
p(r)
Plasmodium falciparum Lipocalin pair distance distribution function Rg: 3.3 nm 0 Dmax: 10.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Plasmodium falciparum Lipocalin SASREF MX model
Plasmodium falciparum Lipocalin SASREF MX model

log I(s)
 s, nm-1

log I(s)
 s, nm-1

log I(s)
 s, nm-1

log I(s)
 s, nm-1

Synchrotron SAXS data were measured from recombinant Plasmodium falciparum lipocalin protein through a concentration series using a standard batch setup as well as in-line size-exclusion chromatography SAXS (SEC-SAXS). All samples were reconstituted in 20 mM Tris pH7.5, 150 mM NaCl, 5% v/v glycerol. The data were collected on the EMBL P12 beam line at PETRA III (Hamburg, Germany) equipped with Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). For the batch measurements, the sample concentrations were: 10, 5, 2 and 1 mg/ml (20 successive 0.050 second frames were collected for each sample at 10°C, in addition to the corresponding matched solvent blank). The data were normalized to the intensity of the transmitted beam and radially averaged and the scattering of the solvent-blank was subtracted to generate the SAXS data displayed in this entry. The primary SAXS data displayed in this entry (top) and the corresponding structural parameters (Rg, p(r), normalized Kratky plot, etc) refer to the 10 mg/ml sample. The additional sample concentration data are represented by the tetramer-dimer equlibrium model fits to the data, and are made available in the full-entry zip-archive. The SEC-SAXS data (fit) is displayed at the bottom of the page. The SEC parameters were as follows: A 75 μl sample at 10 mg/ml was injected at a flow rate of 0.35 ml/min onto a GE Superdex 200 Increase 10/300 column at 20°C. 56 successive 1 second frames were collected through the major SEC-elution peak and processed using CHROMIXS that included the subtraction of an appropriate solvent blank measured from the protein-free column eluate. The Rg-correlation through the SEC-peak is included in the full-entry zip archive.

The models displayed in this entry fit the concentration series and SEC-SAXS data as a concentration-dependent tetramer-dimer equilibrium. The volume fraction of the tetramer decreases systematically as the sample concentration ins decreased, or via dilution through the SEC column (evaluated using SASREFmx in P222 (tetramer) P2 (dimer) symmery). The volume fractions of tetramer are: 10 mg/ml, 0.78; 5 mg/ml, 0.66; 2 mg/ml, 0.44; 1 mg/ml, 0.24 and for SEC, 0.15. Additional SASREFmx models, and their associated volume fractions (that do not differ significantly to the models and volume fractions reported here) are included in the full-entry zip archive.

Plasmodium falciparum Lipocalin (PfLipocalin)
Mol. type   Protein
Organism   Plasmodium falciparum
Olig. state   Tetramer
Mon. MW   22.2 kDa
 
UniProt   Q8I2Q0 (22-217)
Sequence   FASTA
 
PDB code   6TLB