Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15 N and 13 C, opened the door to detailed analyses of macromolecular structure, dynamics and interactions of smaller macromolecules (< ~25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes, and provide a perspective on the wide range of applications of NMR to biochemical problems.Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15 N and 13 C, opened the door to detailed analyses of macromolecular structure, dynamics and interactions of smaller macromolecules (< ~25 kDa). Work on these proteins and nucleic acids has been very fruitful and allowed us to learn much about structurefunction relationships, but is inherently limitied, as the majority of macromolecular complexes of biochemical interest are significantly larger than 25 kDa. Indeed, although much can be learned by examining macromolecules in isolation, mechanistic insights are often only gained upon studying functional higher-order assemblies with partner molecules.NMR studies of large molecules and complexes are complicated by the increased linewidths associated with slower tumbling, and the spectral overlap from the large number of unique signals. Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude (for recent reviews, see (1,2)). Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes, and provide a perspective on the wide range of applications of NMR to biochemical problems. Overcoming Size Limitations: Narrow Lines and Simple SpectraThe slow tumbling of larger macromolecules in solution leads to faster relaxation of transverse magnetization (short T 2 ) due to enhanced spin-spin interactions. One simple, albeit limited, solution to this problem is to increase the overall molecular tumbling rate by recording NMR spectra at elevated temperatures. This can be highly effective for thermostable macromolecules, with the caveat that behavior at physiological temperatures should be † Authors supported by grants from the National Science Foundation (MCB-0092962) and National Institutes of Health (GM067807) *Contact information: foster.281@osu.edu, 614-292-1377, FAX: 614-292-6773. NIH Public Access [3][4][5][6]. Another ingenious approach to reduce tu...

International audienceSometimes less is more: [13C1H3]methyl isotopomers can be biosynthetically incorporated specifically into the pro-S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for large protein assemblies

Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.