Metabolomics is an important component of systems biology research in biology and biomedicine. Two major technologies are widely used in metabolomics research, mass spectrometry and NMR spectroscopy. Both have their own strengths and weaknesses. Recently, LC-MS has gained in popularity, thanks largely to its high sensitivity and ability to detect 10s of thousands of features. In this talk, I will highlight some of the unique strengths of NMR metabolomics, most notably approaches to study metabolic dynamics in real-time in cells or microorganisms. We call this continuous in vivo metabolism by NMR (CIVM-NMR). I will also discuss the difficulty that the entire field faces in confident metabolite identification and will present recent approaches to better combine NMR with LC-MS and computational chemistry to improve compound identification.

Judge, M. T., Wu, Y., Tayyari, F., Hattori, A., Glushka, J., Ito, T., Arnold, J., and Edison, A. S. (2019) Continuous in vivo Metabolism by NMR, Front Mol Biosci 6, 26.  

Edison, A. S.; Colonna, M.; Gouveia, G. J.; Holderman, N. R.; Judge, M. T.; Shen, X.; Zhang, S. NMR: Unique Strengths That Enhance Modern Metabolomics Research. (2021) Anal Chem 93 (1), 478-499.

Our lives are intimately linked to Earth’s 24-hour solar cycle by circadian clocks that coordinate physiology and behavior into rhythms that coincide with the day/night cycle. Recent studies of the genetic basis of morning lark and night owl behavior in humans have identified inherited alleles that alter the intrinsic timing of circadian rhythms. Here, I’ll describe our discovery of the mechanism by which morning lark alleles regulate clock protein dynamics and activity to shorten the human circadian clock by ~4 hours. NMR spectroscopy played a particularly powerful role in this project by identifying how sequential phosphorylation of a multi-serine cluster in the clock protein PERIOD generates feedback inhibition of the major clock kinase, CK1δ, to control clock timing. [ slides ]

HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations. There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed. [ slides ]

Over 300 million years spiders have evolved to produce six different silks and one glue-like substance. Spider silks are comprised almost entirely of protein and are used for a diverse range of applications including web construction, egg case production and wrapping prey. The silks vary dramatically in their mechanical and physical properties with the major ampullate silk (dragline) exhibiting a strength that exceeds steel by weight and a toughness greater than Kevlar while, flagelliform silk has an elasticity comparable to rubber. Our lab is focused on understanding the molecular structure and dynamics of the proteins that comprise the various spider silk fibers with MAS solid-state NMR. It is the folded structures and hierarchical organization of these proteins that imparts spider silks their impressive yet diverse mechanical and physical properties. Our research team has been developing and applying SSNMR to probe secondary structure, hydrogen-bonding, side chain dynamics, and oligomeric protein assembly all of which are crucial to understanding spider silk formation and the resulting fiber properties. Recently, we have focused on using solution NMR to understand the protein-rich fluid within the various silk producing glands to investigate the conformational structure and dynamics prior to fiber formation and determine the important biochemical triggers responsible for converting this hydrogel-like protein solution to fibers with unparalleled, yet diverse mechanical properties. It is our belief that a better fundamental understanding of spider silk protein structure and assembly process will accelerate the ability to mimic and reproduce similar biologically inspired materials in the lab.  [ slides ]

In an effort to develop novel therapeutics to complex biological targets, many new drug modalities that extend beyond traditional small molecule drugs have gained popularity within the drug discovery landscape. Amongst these, peptide-based drugs offer an attractive means of inhibiting protein:protein interactions of featureless protein surfaces and constitute ~10% of new drug approvals in the past 2 years. The hybrid features of peptido-mimetic drugs make analytical and structural characterization using standard small molecule or protein techniques difficult, and a robust analytical toolkit is needed. Herein, we describe several NMR based applications to the characterization of conformationally constrained cyclic peptidomimetic compounds including: 1) concentration determination and purity workflows, 2) triaging of well- behaved single conformer compounds by 1D proton spectral screening, 3) qualitative structural characterization for medium throughput analysis and 4) generation of full 3D structural ensembles on well behaved drug leads. These methods have been applied to address numerous questions in the early discovery process.  [ slides ]

Summer is over, but that doesn’t mean we have to constantly be working.  This is especially true in NMR, where that D1 delay is critical for our spins to relax back to equilibrium prior to collecting data again.   This talk will cover a few different ways of determining T1 to optimize that delay.  And then take a closer look at the various NOAH experiments that aim to get the most information out of that single delay.  [ slides ]

An automated fragmentation quantum mechanics/molecular mechanics approach (AFNMR) has shown promising results in chemical shift calculations for biomolecules. Trends in chemical shift are stable with regards to change in density functional or basis sets, and the use of the small “pcSseg-0” basis, which was optimized for chemical shift prediction, opens the way to more extensive conformational averaging, which can often be necessary, even for fairly well-defined structures. There have also been recent advances in the use of machine learning approaches to develop empirical connections to structure. I will discuss prospects for using both types of calculations to gain insights into biomolecular structure and dynamics.  [ slides ]

In this presentation I will review advances in the detection of surfaces and interfaces using solid-state NMR and how these techniques have been applied in materials science. In particular, I will discuss NMR signal enhancement using cross-effect dynamic nuclear polarization (DNP), as well as methods for generating non-equlibrium electron spin polarization optically with nitrogen-vacancy (NV) centers in diamond and designer chromophore-radical systems.  [ slides ]

Recent advances in structural biology have dramatically expanded the scope of proteins and assemblies that are now amenable to structural analysis. Yet, many biological systems display dynamics and undergo transformations that have been difficult to capture experimentally, especially in complex or native settings. In this context, my group has been developing NMR-based methodologies to describe such challenging systems and to pave the way to the next exciting structural biology frontier, the cellular environment. Here, I will present our progress on two fronts: 1) Development of NMR methodologies to describe the molecular basis of protein phase transitions from the liquid to the gel and solid states. In particular, I will focus on the transitions displayed by heterochromatin protein 1α (HP1α), a key player in gene regulation and chromatin organization. 2) Development of sensitivity-enhanced NMR approaches suitable for the cellular environment. Our work is based on a methodology called dynamic nuclear polarization (DNP) which can transfer polarization from electron to nuclear spins and thus increase nuclear signals by several orders of magnitude. For this purpose, we develop small molecule DNP polarization agents that contain unpaired electron spins and that can be targeted selectively to a protein of interest. These agents have allowed us to obtain NMR structural data of tiny amounts of proteins (1-5 μg) and to study the  polarization transfer mechanisms in targeted DNP experiments. [ slides ]