Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

Abstract

Abstract:The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N-and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

Interaction of the GTPase Elongation Factor Like-1 with the Shwachman-Diamond Syndrome Protein and Its Missense Mutations

“…The maximum value of 1.1 at 3 for q * R g corresponds to a globular and compact protein, similar to the bovine serum albumin (BSA) protein used as a standard in the experiment. The Sdo1 curve displayed a hyperbolic plateau suggesting a highly flexible protein, as has been previously described for the Archaeoglobus fulgidus SBDS [23]. Interestingly, deletion of domain 1 in Sdo1 resulted in a Kratky plot characteristic of a compact protein.…”

“…The three- and four-state models comprise conformations with similar R g and similar relative abundance that differ from those described in the 2-state model by the presence of an extended conformation (Supplementary Table S2). We selected the three-state models, since a similar flexibility analysis for the A. fulgidus SBDS using the program EOM also predicted such a conformational ensemble [23], and contained the most populated conformations at 27–29 Å. This ensemble comprised a ‘compact’ conformation with an R g of 24 Å, a ’stretched’ one with R g of 28 Å, and a ‘relaxed’ conformation having an R g of 30.4 Å.…”

A Comparative Molecular Dynamics Study of Selected Point Mutations in the Shwachman–Bodian–Diamond Syndrome Protein SBDS

“…In other words, the picture emerging from these data suggests that when involved in strong interactions with domain I, domain III might not be available for other interactions; including the interaction with EFL1. Our data also point out that a dynamical equilibrium between the two distinct conformations (open and closed) is crucial to ensure a proper SBDS function, in full agreement with our previous observations [ 23 , 24 ]. Importantly, all the investigated mutants, irrespective of the considered starting conformation (open or closed), tend to assume an intermediate state, as evident from the computed ∆σ.…”

“…Open/closed transitions of the SBDS conformation occur spontaneously in physiological solutions, hence the protein pool should be represented in percentages of closed and open structures [ 23 , 24 ]. Introducing mutations that affect the protein structure stability may alter the population of these conformations.…”

Cation folding and the thermal stability limit of the ionic liquid [BMIM+][BF4−] under total vacuum

“…These two normal distributions depict sets of conformations categorized as folded and unfolded (extended) conformations. Systems with more, longer chains, such as proteins 49,50 and polymers, 51,52 show these bimodal distributions in R g . At the mean values of the distributions, the difference in the mean R g is small (0.17Å), and it is difficult to determine if a specic conformation belongs to a specic distribution because the distributions overlap.…”