Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques.

Linda Cerofolini,Enrico Ravera,Vito Calderone,Marco Fragai,Ludovic Renault,Christoph A Diebolder

doi:10.3390/biom9080370

Abstract

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.

Highlights

Biomolecules 2019, 9, 370 same time, NMR spectroscopy has undergone technical and methodological advancements allowing for the characterization of difficult systems such as very large or transient protein complexes, fibrils, and protein embedded in matrices [12,13,14,15]
To characterize these large PEGylated proteins/complexes, we relied upon a combined use of solution NMR and X-ray crystallography and solid-state NMR experiment collected on the conjugated l-asparaginase
Was collected to assign the protein regions retaining a large mobility; (iv) 2D and 3D solid-state NMR (SSNMR) spectra were collected on crystalline and PEGylated samples of the protein to complete an extensive resonance assignment, integrating the data collected in solution; (v) structural restraints are derived from 13 C-13 C

Summary

Introduction

We have shown that the integration of NMR and X-ray crystallography allows for the obtainment of an experimental and reliable structure of a large PEGylated protein [33]. The comparison of 13 C-13 C two-dimensional SSNMR spectra collected on some native proteins and on their PEGylated forms proves that in all cases 3-D structure of the protein core is preserved after conjugation with PEG [37].

Results

Conclusion