Emerging applications of small angle solution scattering in structural biology

Small angle solution X‐ray and neutron scattering recently resurfaced as powerful tools to address an array of biological problems including folding, intrinsic disorder, conformational transitions, macromolecular crowding, and self or hetero‐assembling of biomacromolecules. In addition, small angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy, and other structural methods to aid in the structure determinations of multidomain or multicomponent proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation, or X‐ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for characterizing the organizations of two‐component systems such as a nucleoprotein or a lipid‐protein assembly. Time‐resolved small and wide‐angle solution scattering to study biological processes in real time, and the use of localized heavy‐atom labeling and anomalous solution scattering for applications as FRET‐like molecular rulers, are amongst promising newer developments. Despite the challenges in data analysis and interpretation, these X‐ray/neutron solution scattering based approaches hold great promise for understanding a wide variety of complex processes prevalent in the biological milieu.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Hydration of proteins: SAXS study of native and methoxy polyethyleneglycol (mPEG)-modified L-asparaginase and bovine serum albumin in mPEG solutions

Two mPEG-modified globular proteins [mPEG: methoxy poly(ethylene glycol)], and their native unmodified forms, were examined by small-angle x-ray scattering to evaluate the extent of their surface hydration. The effects of free and protein-bound mPEG on the hydration shell were modeled with discrete electron density profiles. We show that an mPEG-depleted layer can account for the decrease in the measured radius of gyration R(g) from 34.1 to 31.1 A in native L-asparaginase, and from 32.4 to 31.0 A in native bovine serum albumin (BSA) in mPEG-containing solvents. For mPEG-modified proteins in mPEG-free solvents, we attribute the observed increase in the R(g) over that of the native proteins (approximately 3% in L-asparaginase, and 10% in BSA) to the presence of mPEG on the protein surface. The R(g) of the mPEG-modified proteins in mPEG solutions generally decrease with mPEG concentration. Relative to the corresponding unmodified protein, this decrease in R(g) is much larger in BSA (from 35.6 to 31.2 A) but much smaller (from 34.9 to 34.3 A) in L-asparaginase. From these studies, the thickness of the hydration layer around L-asparaginase and BSA is estimated to be approximately 15 A. Exclusion of polyols from the protein domain could be related to the presence of the hydration shell around the protein.     

Monitoring the crystallization of amylose–lipid complexes during maize starch melting by synchrotron x‐ray diffraction

Most starch granules exhibit a natural crystallinity, with different diffraction patterns according to their botanical origin: A‐type from cereals and B‐type from tubers. The V polymorph results essentially from the complexing of amylose with compounds such as iodine, alcohols, or lipids. The intensity and nature of phase transitions (annealing, melting, polymorphic transitions, recrystallization, etc.) induced by hydrothermal treatments in crystalline structures are related to temperature and water content. Despite its small concentration, the lipid phase present mainly in cereal starches has a large influence on starch properties, particularly in complexing amylose. The formation of Vh crystalline structures was observed by synchrotron x‐ray diffraction in native maize starch heated at intermediate and high moisture contents (between 19 and 80%). For the first time, the crystallization of amylose–lipid complexes was evidenced in situ by x‐ray diffraction without any preliminary cooling, at heating rates corresponding to the usual conditions for differential scanning calorimetry experiments. For higher water contents, the crystallization of Vh complexes clearly occurred at 110–115°C. For intermediate water contents, mixed A + Vh (or B + Vh for high amylose starch) diffraction diagrams were recorded. Two mechanisms can be involved in amylose complexing: the first relating to crystallization of the amylose and lipid released during starch gelatinization, and the second to crystalline packing of separate complexed amylose chains (amorphous complexes) present in native cereal starches. © 1999 John Wiley & Sons, Inc. Biopoly 50: 99–110, 1999