Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g. states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments, as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 minutes.

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here we report on the results of an investigation of an intrinsically disordered 188-residue fragment (IDP) of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline SH3 binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1, a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a non-canonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during HCV infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

Selective isotopic unlabeling of proteins can provide important residue-type information as well as reduce congestion of NMR spectra. However, metabolic scrambling often complicates the final isotope-labeling pattern. Here, an array of metabolic precursors is used to perform robust, residue-specific unlabeling of proteins. The resulting isotopic-labeling patterns are predictable and nicely complement NMR experiments that differentiate residue types. This approach has widespread applications, but it is particularly relevant for proteins that lack sequence complexity or a defined tertiary structure.

Probing protein structure, dynamics, and interaction surfaces by NMR requires initial backbone resonance assignment. The protocol for this step has been progressively developed in the last 15 years to provide robust assignments. However, even in the case of favorable conditions (high field magnets and cryogenically cooled probes, small globular proteins, high sample concentration), the assignment step generally takes several days of data collection and analysis, thus precluding studies of unstable proteins and limiting high-throughput applications. Recently, we have introduced the BATCH strategy for fast protein backbone resonance assignment. BATCH benefits from the combination of several tools (BEST/ASCOM/Targeted-Sampling/COBRA/HADAMAC) for time-optimized and highly automated NMR data acquisition, processing, and analysis. In this chapter, we discuss the individual steps of the BATCH method and describe its practical implementation to obtain the backbone resonance assignment of small globular proteins in a few hours of time.

An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.

It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.

Heparan sulfate (HS), a polysaccharide of the glycosaminoglycan family characterized by a unique level of complexity, has emerged as a key regulator of many fundamental biological processes. Although it has become clear that this class of molecules exert their functions by interacting with proteins, the exact modes of interaction still remain largely unknown. Here we report the engineering of a (13)C-labeled HS-like oligosaccharide with a defined oligosaccharidic sequence that was used to investigate the structural determinants involved in protein/HS recognition by multidimensional NMR spectroscopy. Using the chemokine CXCL12α as a model system, we obtained experimental NMR data on both the oligosaccharide and the chemokine that was used to obtain a structural model of a protein/HS complex. This new approach provides a foundation for further investigations of protein/HS interactions and should find wide application.

Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A's multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential resonance assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in (1)H-(15)N correlation spectra. Our assignment reveals the presence of several segments with high propensity to form α-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.

Band-selective radiofrequency (rf) pulses provide powerful spectroscopic tools for many biomolecular NMR applications. Band-selectivity is commonly achieved by pulse shaping where the rf amplitude and phase are modulated according to a numerically optimized function. This results in complex spin evolution trajectories during the pulse duration. Here we introduce simplified representations of a number of shaped pulses, commonly used in biomolecular NMR spectroscopy. These simple schemes, consisting in a suite of free evolution delays and ideal rf pulses, reproduce astonishingly well the effect on a scalar-coupled hetero-nuclear two-spin system. As a consequence, optimal use of such pulse shapes in complex multi-pulse sequences becomes straightforward, as demonstrated here for the example of longitudinal-relaxation-enhanced BEST-HSQC and BEST-TROSY experiments. Applications of these optimized pulse sequences to several proteins in the size range of 8-21 kDa are shown.

Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.