Search

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.

International audience; Mechanical resilience of bone tissue decreases with age. The ability to comprehensively probe and understand bone properties could help alleviate this problem. One important aspect of bone quality that has recently been made evident is the presence of dilatational bands formed by osteocalcin (OC) and osteopontin (OPN), which contribute to fracture toughness. However, experimental evidence of the structural role of these two proteins at the organic-mineral interface in bone is still needed. Solid state nuclear magnetic resonance (SSNMR) is emerging as a useful technique in probing molecular level aspects of bone. Here, we present the first SSNMR study of bone tissue from genetically modified mice lacking OC and/or OPN. Probing the mineral phase, the organic matrix and their interface revealed that, despite the absence of OC and OPN, the organic matrix and mineral were well preserved, and the overall exposure of collagen to hydroxyapatite (HA) nanoparticles was hardly affected. However, the proximity to the HA surface was slightly increased for a number of bone components including less abundant amino acids like lysine, suggesting that this is how the tissue compensates for the lack of OC and OPN. Taken together, the NMR data supports the recently proposed model, in which the contribution of OC-OPN to fracture toughness is related to their presence at the extrafibrillar organic-mineral interfaces, where they reinforce the network of mineralized fibrils and form dilatational bands. In an effort toward further understanding the structural role of individual amino acids of low abundance in bone, we then explored the possibility of specific 13C enrichment of mouse bone, and report the first SSNMR spectra of 97% 13C lysine-enriched tissue. Results show that such isotopic enrichment allows valuable molecular-level structural information to be extracted, and sheds light on post-translational modifications undergone by specific amino acids in vivo.

Heparin and HS (heparan sulfate) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS binding to VEGF (vascular endothelial growth factor) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that the VEGF binding affinity likely depends on the specific structural features of these oligosaccharides, including their degree of sulfation, sugar-ring stereochemistry and conformation. Notably, the unique 3-O-sulfo group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue-specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.