Despite the abundance of collagen in humans, studying its molecular interactions has proven remarkably difficult using conventional biophysical techniques. But as shown by a research team in Lucknow, India, this barrier to research is now being lifted thanks to sensitivity enhancements for solid-state NMR. The team has shed new light on the reasons for the structural stability of collagen at molecular level and made progress in tackling bone-degenerative conditions such as osteoporosis.
Limitations of existing biophysical methods
Collagen is the most abundant protein in mammals and humans, and is largely found in connective tissues, such as bones, cartilage, tendons, ligaments and skin. Within these structures, collagen is largely found within the extracellular matrix (ECM), which as well as providing structural integrity, has numerous physiological roles, including storage of nutrients, delivery of growth factors and tissue repair.
Studying ECM is therefore important to understand various properties of the connective tissue itself – including its load-bearing capacity, tensile and shear strength, and shock absorption characteristics. However, the properties of collagen depend upon the presence of, and interactions with, other components and so the majority of studies on collagen have been carried out using extracted collagen, model peptides, and molecular dynamics simulations. The limitations of using ex vivo models have meant that the reasons behind the structural stability of various collagen-containing biomaterials – including bones and cartilage – is still a mystery.
A team led by Dr. Neeraj Sinha at the Centre of Bio-Medical Research (CBMR), Lucknow, India, is using nuclear magnetic resonance (NMR) spectroscopy to obtain a molecular-level understanding of collagen-mediated changes in bone and cartilage, leading to work towards improved patient outcomes in conditions such as osteoporosis.
Investigating collagen in its native state
To understand how NMR is able to assist in this project, it first helps to appreciate the structure of collagen. Collagen largely consists of a repeating sequence of three amino acids – glycine, proline and hydroxyproline – with individual strands organised into a triple-helix structure. This is primarily stabilised by water-mediated hydrogen bonds and a stereoelectric effect resulting from the hydroxyproline ring.
The role of collagen in various tissues depends upon interactions with other molecules. In bones, citrate plays a key role in improving the deposition of hydroxyapatite (one form of calcium phosphate), and thus contributes to bone structure and strength. In cartilage, proteoglycans comprising glycosaminoglycan (GAG) chains attached to a core protein are a key component of the ECM and, because of their water-attracting nature, are crucial in providing cartilage with its lubricating and shock-absorbing functions. They also govern the activity of chondrocytes (the cells responsible for cartilage formation) and the assembly of the ECM itself.
Understanding these interactions at molecular level can provide better insights into their mechanism of action, and therefore of the consequences for conditions such as osteoporosis and osteoarthritis. But doing this within the native molecular environment of the collagen is highly challenging for most biophysical techniques, because of the complex, heterogeneous structure of the ECM. For example, X-ray spectroscopy, Fourier-transform infrared, Raman spectroscopy, micro-computed tomography and atomic force microscopy have all been used to determine structural details of collagen in its extracted state, but these methods cannot detect the dynamics and orientation-dependent molecular interactions in the native state within the bone.
NMR spectroscopy offers a way forward, thanks to its ability to directly probe atomic-level interactions in highly complex, heterogeneous systems, with solid-state NMR being of particular value.
What is solid-state NMR?
Solid-state NMR spectroscopy is a set of experimental approaches, enabled by specialized hardware, that allows various kinds of solid, semi-solid or partly immobilized samples to be studied. This is in contrast to conventional NMR, which uses solution-phase samples.
One challenge of solid-state NMR is achieving sufficiently narrow spectral lines, which, as well as enabling easier interpretation of 1D spectra, opens the door to the 2D experiments that underpin most biochemical studies. In solution-phase spectra of small molecules, line width is not a problem because the fast, isotropic tumbling of the molecules effectively ‘averages out’ the signals. But larger proteins and assemblies, or those that are bound to a substrate, tumble too sluggishly, or not at all, for this to be effective.
Various approaches – of which magic-angle spinning (MAS) is the most important and versatile – overcome this problem and allow NMR to be applied to many types of non-solution-phase biological samples. These have historically focused on lipid bilayer membranes and proteins within them, as well as protein misfolding and aggregation disorders. More recently, solid-state NMR has been used to study biological assemblies, including non-amyloid protein aggregates, protein–protein complexes and viral capsids, as well as the native-state biological solids that are a focus of Dr. Sinha’s team’s work.
Boosting the sensitivity of solid-state NMR for collagen analysis
Even with the use of solid-state NMR, difficulties are encountered because NMR typically relies on improving signal-to-noise ratios (and hence sensitivity) using isotopic enrichment – a route that is not possible when studying biomolecular systems in their native state. This is especially a problem in bone, where concentrations of organic molecules are already low.
To tackle these limitations and enhance NMR sensitivity, Dr. Sinha’s team uses three approaches:
• High-frequency spinning at the magic angle of 54.7° mimics the tumbling of molecules in solution and leads to an improvement in peak shape for solid samples, suppressing the line broadening that would otherwise result from interactions involving 1H nuclei.
• Using MAS cryoprobe technology with cold preamplifier and electronics at about –170 °C reduces background noise and allows sensitivity to be improved 3-4-fold without additional line broadening.
• Dynamic nuclear polarization uses microwaves to transfer spin polarization from electrons to nuclei, increasing the response from the nuclei and improving the signal-to-noise ratio. The team used this approach in some experiments to study 15N nuclei, which has very low natural occurrence and therefore an intrinsic low sensitivity.
Using these methods, Dr. Sinha’s team has been able to probe the interactions of collagen with other components of the ECM using natural-abundance isotopes and shed light on the reasons for its structural stability.
Probing molecular interactions and structural features of collagen
In the latest piece of work, Dr. Sinha’s group investigated the cortical femora bone and articular cartilage of a goat. In each case, about 60 mg of powdered sample, mixed (if needed) with a biradical polarizing agent, was packed in an MAS rotor, ready for analyses of the correlations between their 1H, 13C and 15N spectra using a 600 MHz NMR system.
This simple preparation procedure is an advantage for biological samples, which can be negatively affected by harsh conditions or lengthy processing. Observed 13C resonances from the backbone of the collagen proteins under investigation were similar, whether or not the polarising agent and low-temperature conditions were used. This confirmed that these experimental modifications had not affected the structural integrity of the samples.
The team probed the collagen–citrate interactions in bone, and the collagen–GAG interactions in cartilage, and found that in both cases there were a number of short-range and long-range interactions with amino acids in the collagen protein (Figure 1). In addition to these experiments, the researchers used dynamic nuclear polarization to boost sensitivity for the 15N nucleus, enabling them to carry out 2D experiments that would otherwise have been impossible. These showed significant differences between the structure of collagen in bone and in cartilage, in terms of the molecular arrangement of the backbone and side-chain residues.
Figure 1: Natural-abundance 2D NMR spectrum showing the correlation between the signals from 13C nuclei (x-axis) and 1H nuclei (y-axis) in the ECM of bone. The expanded region (below) shows the molecular interaction between citrate and certain amino acids in collagen, shown by the dotted lines. Reprinted with permission from ref. 2. Copyright 2021 American Chemical Society.
Conclusion
The conclusion from the work of Dr. Sinha and his group is that citrate and GAG are intimately involved in interactions with collagen in bone and cartilage respectively. In bone, the research suggests that collagen protein in the bone ECM may be involved in citrate homeostasis in the bone metabolic pathway, while in cartilage, it infers that GAGs may have a role in maintaining the physiochemical properties of the ECM.
Combined with the collagen structural studies enabled by dynamic nuclear polarization this allows researchers to better understand the mechanisms involved in the structural stability of collagen within the ECM. Such insights are only possible thanks to the sensitivity enhancements now accessible with modern solid-state NMR equipment, which allows complex nucleus–nucleus correlation experiments to be performed on native samples in the solid state.
The consequence of this better understanding will ultimately be tools that will guide the development of prevention and care therapies for bone and cartilage disorders such as osteoporosis and osteoarthritis – a need that will undoubtedly continue to increase in societies with aging populations.
For more information on Bruker’s NMR solutions, please visit: https://www.bruker.com/en/products-and-solutions/mr/nmr.html
About Bruker
Bruker is enabling scientists to make breakthrough discoveries and develop new applications that improve the quality of human life. Bruker’s high-performance scientific instruments and high-value analytical and diagnostic solutions enable scientists to explore life and materials at molecular, cellular and microscopic levels. In close cooperation with our customers, Bruker is enabling innovation, improved productivity and customer success in life science molecular research, in applied and pharma applications, in microscopy and nanoanalysis, and in industrial applications, as well as in cell biology, preclinical imaging, clinical phenomics and proteomics research and clinical microbiology.
Author: Dr. Neeraj Sinha
Author: Dr. Neeraj Sinha
