Designing and defining dynamic protein cage nanoassemblies in solution.

Central challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. We created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. To generate a phase diagram associating structure and assembly, we developed force plots that measure dissimilarity among multiple SAXS data sets. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. These methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes.

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.

High-throughput SAXS for the characterization of biomolecules in solution: a practical approach.

The recent innovation of collecting X-ray scattering from solutions containing purified macromolecules in high-throughput has yet to be truly exploited by the biological community. Yet, this capability is becoming critical given that the growth of sequence and genomics data is significantly outpacing structural biology results. Given the huge mismatch in information growth rates between sequence and structural methods, their combined high-throughput and high success rate make high-throughput small angle X-ray scattering (HT-SAXS) analyses increasingly valuable. HT-SAXS connects sequence as well as NMR and crystallographic results to biological outcomes by defining the flexible and dynamic complexes controlling cell biology. Commonly falling under the umbrella of bio-SAXS, HT-SAXS data collection pipelines have or are being developed at most synchrotrons. How investigators practically get their biomolecules of interest into these pipelines, balance sample requirements and manage HT-SAXS data output format varies from facility to facility. While these features are unlikely to be standardized across synchrotron beamlines, a detailed description of HT-SAXS issues for one pipeline provides investigators with a practical guide to the general procedures they will encounter. One of the longest running and generally accessible HT-SAXS endstations is the SIBYLS beamline at the Advanced Light Source in Berkeley CA. Here we describe the current state of the SIBYLS HT-SAXS pipeline, what is necessary for investigators to integrate into it, the output format and a summary of results from 2 years of operation. Assessment of accumulated data informs issues of concentration, background, buffers, sample handling, sample shipping, homogeneity requirements, error sources, aggregation, radiation sensitivity, interpretation, and flags for concern. By quantitatively examining success and failures as a function of sample and data characteristics, we define practical concerns, considerations, and concepts for optimally applying HT-SAXS techniques to biological samples.