Ronald A. Venters

General theory of polycrystalline ENDOR patterns. g and hyperfine tensors of arbitrary symmetry and relative orientation

Abstract A general formulation is presented for the ENDOR spectra of a randomly oriented, polycrystalline (powder) S = 1 2 paramagnet, assuming slow cross-relaxation, g anisotropy domination of the EPR spectrum, arbitrary symmetries and relative orientations of g and hyperfine tensors, and δ-function EPR and ENDOR component lineshapes. Calculations for simple, archetypical types of centers have been presented, as well as selected calculations showing the full generality of the method. In particular, at g values where the ENDOR spectra arise from multiple orientations of the paramagnet (powder-type spectra) it was found that the ENDOR shape functions, N ( A ) g , show two divergences and no steps when the g and A tensors are coaxial; noncoaxiality can introduce subsidiary maxima and can cause the occurrence of as many as four additional divergences.

High-level 2H/13C/15N labeling of proteins for NMR studies

SummaryThe protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties.

Radial sampling for fast NMR: Concepts and practices over three decades.

For almost as long as three- and four-dimensional NMR experiments have been used, NMR spectroscopists have been devising ways to speed them up. Indeed, the publication that is often cited as the very first to demonstrate a 3-D NMR experiment commented, “it has been thought that…high-resolution 3-D NMR experiments are impracticable because of huge data matrices and long measurement times,” but went on to “suggest a technique for reduction of data matrices” using selective excitation pulses [1]. Although both spectrometers and computers have advanced considerably since this 1987 publication, somewhat changing the definitions of huge and long, the fundamental problem of measurement time continues to limit the experiments that can be carried out in practice using conventional multidimensional NMR methodology, and significant effort is still devoted to alleviating this restriction. The problem arises from the very nature of Fourier transform (FT) NMR, which involves the systematic sampling of a signal over time followed by the calculation of the spectrum using the FT [2, 3]. Traditionally, an n-dimensional (n-D) experiment is obtained through the sampling of the time domain on a complete n-D Cartesian grid; since the number of points in an n-D grid grows exponentially with the number of dimensions n, the measurement time needed to record the experiment becomes considerable even for small n. Yet NMR spectra are generally only sparsely populated with signals, suggesting that there is no statistical need for so many observations (other than signal accumulation for sensitivity in some cases), and that a suitable alternative approach to sampling and/or processing might significantly reduce the time requirement, while generating the same or more spectral information. Two trends in biomolecular NMR research have given particular impetus to these efforts: the drive for increased throughput in studies of small proteins—for example in structural genomics—which requires running today’s routine experiments more quickly, and the increasing attention given to large and challenging systems, which require more dimensions and higher resolution than conventional experiments can offer. A variety of methods have been introduced in these efforts to reduce the amount of data needed for multidimensional NMR, but a surprising number of them share in common that they sample the indirect dimensions of the time domain along radial spokes. The measurement of a radial spoke simply means collecting data samples along a line in the time domain that passes through the origin. If multiple radial spokes are sampled, the resulting dataset is equivalent to recording the NMR experiment in cylindrical coordinates or their higher-dimensional equivalent (cylindrical rather than spherical because the directly observed dimension is always sampled conventionally). The potential advantage of this arises from the fact that one can arrange the radial sampling points so as to obtain higher resolution information than with conventional Cartesian sampling, for the same or a smaller number of samples. Depending on the processing method used to extract spectral information from the data, this approach may lead to artifacts or ambiguities—but it also has the potential to provide complete spectral information in considerably less time than required for conventional NMR. The purpose of this article is to review the long history of radial sampling in NMR, from its initial introduction in the “accordion spectroscopy” experiments, through reduced-dimensionality and G-matrix Fourier Transform (GFT) spectroscopy, and on to the projection spectroscopy and projection-reconstruction techniques. Because all of these methods share a common mathematical foundation—despite their sometimes differing vocabularies—we first explain these underlying concepts. We then continue with a chronological survey of the different approaches, describing how they were developed, how they work and how they have been put to use. Particular attention is given to how these methods can be used to reduce the measurement time of the experiment, including the theoretical basis for the time savings and the practical tradeoffs that can result. It is important to note that while this review describes a number of techniques for reducing NMR measurement time, it does not attempt to describe the many methods that have been introduced recently for that purpose which do not use radial sampling. These include random sampling [4–11], concentric ring or shell sampling [12, 13] and other unconventional sampling approaches (e.g. spiral [14]); filter diagonalization analysis to extract high-resolution information from low-resolution conventionally sampled data [15–17]; the measurement of a spectrum in a single scan through the encoding of the spectroscopic frequency information spatially within the sample [18, 19]; Hadamard encoding to measure signal intensities at a small number of directly excited frequencies [20–22]; covariance spectroscopy, which enhances the resolution in the indirect dimensions through a statistical symmetrization with the directly observed dimension [23–26]; and the “minimal sampling” procedure, which involves calculating the possible correlations between the signals on the “first planes” of a multidimensional experiment and resolving any ambiguities by measuring a single additional sampling point [27, 28]. We touch on processing methods such as multidimensional decomposition [7, 29] and maximum entropy reconstruction [30] only to the limited extent that they have been applied to radial sampling experiments. Additionally, we do not discuss methods for reducing experiment time by optimizing the longitudinal relaxation rate to allow a much shorter interscan delay, which could be applicable to any type of sampling [31–34].

Structure, binding interface and hydrophobic transitions of Ca(2+)-loaded calbindin-D(28K).

Calbindin-D28K is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D28K reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of α5 (EF3), α8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D28K provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.

General theory of polycrystalline ENDOR patterns: effects of finite EPR and ENDOR component linewidths

Recently (I) we presented a general method for calculating ENDOR spectra of a randomly oriented polycrystalline (powder) S = 4 paramagnet (2, 3) that has g and hyperfme tensors of arbitrary symmetry and relative orientation. That study (Paper I) laid a foundation for the detailed analysis of polycrystalline ENDOR spectra, as well as for the heuristic categorization of such spectra, much as EPR spectra long have been classified (isotropic, axial, rhombic (4)). To this end the calculations in I used a “double delta function” approach, employing a-function EPR and ENDOR component lineshapes. They showed that polycrystalline ENDOR spectra can exhibit a rich array of features when the static field is set to a g value where the EPR signal arises from a well-defined set of orientations of the paramagnet, rather than a single crystal-type setting. The principal values and relative orientation of a hyperline tensor can be obtained by examining such angle-selected spectra obtained as the observing g value (static field) is moved across the EPR envelope. We now have generalized the analysis to permit full simulations of polycrystalline ENDOR spectra by including finite EPR and ENDOR component linewidths. The calculations presented here show that the two types of features that arise in the double delta-function limit, divergences and maxima, both persist as absorption peaks in the full simulations. These ENDOR peaks broaden equally as the ENDOR component linewidth, W,,, increases, but in general they do not broaden equally as the EPR component linewidth, W,, increases. Instead, the rate at which a peak broadens with W, is related to (&/dg), the rate of change in its ENDOR frequency with observing g value. The essential features of the approach developed in I are first summarized, and then are extended to include EPR and ENDOR component linewidths. When the external field is set to an arbitrary value, Ho, within a a-function EPR envelope, the EPR signal intensity, and thus an ENDOR signal, arises from the selected molecular orientations associated with the curve on the unit sphere, S, comprising points for which the orientation dependent spectroscopic splitting factor, g = g(B, @), has a fixed value defined by the strength of the observing field: gmi, < g = hv/@& < g,,,. H’owever, although g is constant over the locus of points, S, in general the angledependent hyperfine coupling, A = A(& 4), is not, and the ENDOR pattern in general is more complex than a single crystal-like spectrum. The net ENDOR intensity at radiofrequency, Y, is determined by the sum of the probabilities that the

A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

SummaryA 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C,15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.

Calbindin D28K interacts with Ran-binding protein M: identification of interacting domains by NMR spectroscopy

Calbindin D(28K) is an EF-hand containing protein that plays a vital role in neurological function. We now show that calcium-loaded calbindin D(28K) interacts with Ran-binding protein M, a protein known to play a role in microtubule function. Using NMR methods, we show that a peptide, LASIKNR, derived from Ran-binding protein M, interacts with several regions of the calcium-loaded protein including the amino terminus and two other regions that exhibit conformational exchange on the NMR timescale. We suggest that the interaction between calbindin D(28K) and Ran-binding protein M may be important in calbindin D(28K) function.

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins.

SummaryThe perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain 13C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D 15N/15N-separated NOESY, as many main-chain and side-chain 1HN/15N resonances as possible must be assigned. Traditionally, only backbone amide 1HN/15N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain 1HN/15N resonances to side-chain 13C resonances with high sensitivity: NH2-filtered 2D 1H-15N HSQC (H2N-HSQC), 3D H2N(CO)Cγ/β and 3D H2N(COCγ/β)Cβ/α for glutamine and asparagine side-chain amide groups; 2D refocused H(Nε/ζ)Cδ/ε and H(Nε/ζCδ/ε)Cγ/δ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(Nε)Cζ and nonrefocused H(Nε.η)Cζ for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated 13C-/15N-labeled human carbonic anhydrase II (2H-HCA II). Because more than 95% of all side-chain 13C resonances in 2H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain 1HN/15N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain HN protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only 1HN-1HN NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].

The effects of Ca2+ binding on the conformation of calbindin D28K: A nuclear magnetic resonance and microelectrospray mass spectrometry study

Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i). apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer.

NMR Structure of AbhN and Comparison with AbrBN FIRST INSIGHTS INTO THE DNA BINDING PROMISCUITY AND SPECIFICITY OF AbrB-LIKE TRANSITION STATE REGULATOR PROTEINS

Understanding the molecular mechanisms of transition state regulator proteins is critical, since they play a pivotal role in the ability of bacteria to cope with changing environments. Although much effort has focused on their genetic characterization, little is known about their structural and functional conservation. Here we present the high resolution NMR solution structure of the N-terminal domain of the Bacillus subtilis transition state regulator Abh (AbhN), only the second such structure to date. We then compare AbhN to the N-terminal DNA-binding domain of B. subtilis AbrB (AbrBN). This is the first such comparison between two AbrB-like transition state regulators. AbhN and AbrBN are very similar, suggesting a common structural basis for their DNA binding. However, we also note subtle variances between the AbhN and AbrBN structures, which may play important roles in DNA target specificity. The results of accompanying in vitro DNA-binding studies serve to highlight binding differences between the two proteins.