All-Atom Ensemble Modeling to Analyze Small-Angle X-Ray Scattering of Glycosylated Proteins

Abstract

Summary The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small Angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well defined protein structure), all-atom modeling generates models in excellent agreement with the scattering pattern, and reveals the approximate spatial occupancy of the glycan chain in solution. For more complex systems (several glycan chains, or unknown protein substructure), the approach can still provide insightful models, though the orientations of glycans become poorly determined. Ab initio shape reconstructions appear to capture the global morphology of glycoproteins, but in most cases offer little information about glycan spatial occupancy. The all-atom modeling methodology is available as a webserver at http://modbase.compbio.ucsf.edu/allosmod-foxs.

A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140

“…Static light scattering (SLS) and zero-angle intensity from SAXS were used to assess the oligomeric state of each species since these methods have been shown to be accurate for such highly glycosylated proteins (35,46). In addition, dimeric and monomeric gp120 were examined to validate the methods for molecular mass determination (Table 1).…”

“…Molecular masses were calculated for each sample using the zero angle scattering intensity [I(0)] relative to water or RNase A as a standard (45), with adjustments for carbohydrate content as described previously (46). The particle distance distribution function [P(r)] plot generated from GNOM (44) was used for ab initio shape reconstruction using DAMMIN v5.3i (47).…”

“…We note that the uncleaved gp140s examined here were expressed and purified by methods identical to those previously described and in widespread use (25,28). Obtaining accurate molecular mass measurements for glycoproteins is not trivial (46), and it is plausible that the oligomers that are presumed to be trimers based on native PAGE or gel filtration chromatography in many cases are in fact dimerized gp140.…”

The galectin lattice at a glance

“…Mixing experiments with Gal3 and a bivalent N-glycan in vitro have revealed rapid phase transitions at critical molar ratios between Gal3 and ligands (Ahmad et al, 2004); dynamics are similar to that of phase transitions by adaptor proteins in receptor signaling (Li et al, 2012) or stress-induced RNA-protein granules (Han et al, 2012). N-glycans generally extend away from the protein surface and are flexible due to rotational freedom of the glycosidic bonds (Guttman et al, 2013). Multivalent interactions and a flexible geometry contribute to the intrinsic disorder (i.e.…”

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…Also small-angle X-ray/neutron scattering and light scattering provide information on the size and shape of the coated proteins,b ut cannot map the conjugation sites. [28,29] More detailed information on the conjugation sites along the primary sequence of the protein can be achieved by mass spectrometry. [27] However,a lso for mass spectrometry the evaluation of the conjugation degree at the different sites in polydisperse populations of isomers is ac hallenge.R ecently,w eh ave shown that structural information on large PEGylated proteins,s uch as E. coli l-Asparaginase-II (138 kDa, ANSII, see the Supporting Information), can be obtained by solid-state NMR spectroscopy.…”