Jens J. Led

The applicability of the magnetization-transfer NMR technique to determine chemical exchange rates in extreme cases. The importance of complementary experiments

Abstract It is shown that a conventional two-site magnetization-transfer experiment provides an insufficient amount of information to allow a reliable determination of the exchange rate, unless |λ2| ⪢ |λ1|, where λ1 and λ2 are the time constants of the two exponentials involved. This difficulty, which is often met, is further hampered when the linewidths as well as the longitudinal relaxation rates of the two exchanging signals are different, when a perturbation of the noninverted signal occurs, or when the signal intensities are difficult to determine. However, by including a complementary experiment, in which the perturbed and unperturbed signals are interchanged, additional, independent information about the parameters involved is provided, allowing all of the unknown parameters including the exchange rate to be determined in most cases. The significant increase in the applicability of the technique when the complementary experiment is included, as well as the considerable errors that may be introduced when it is omitted, is illustrated by examples of the rate of exchange between the NH and H2O protons in the water/N-methylacetamide system. In addition these experiments demonstrate that exchange rates covering about three orders of magnitude (0.06 to 45 sec−1) can be determined by the method when applied in the form presented here, as exchange rates which are at least seven times slower than the fastest relaxation rate can be evaluated.

Microwave spectra of isotopic cyclobutenes: Molecular structure of cyclobutene

Abstract Microwave spectra of cyclobutene and four monosubstituted isotopic species have been recorded and analyzed, yielding a conventional r s -structure ( C 2v symmetry). The carbon ring is planar with valence angles 94.2° (at the double bond) and 85.8°. The C(1)-C(2) distance is 1.342 A, the C(2)-C(3) distance 1.517 A, and the C(3)-C(4) distance 1.566 A, all of these longer than any acyclic normals.

Quantitative analysis of complicated nonideal fourier transform NMR spectra

Abstract The exact expression for the discrete Fourier transform of a sum of exponentially damped sinusoids is derived, and its applicability for describing the general, discrete Fourier transform spectrum is demonstrated. It is shown that the frequency, the linewidth, the intensity, and the phase of each individual signal can be determined by fitting the derived expression to the real and imaginary part of experimental spectra. The results are not influenced by aliasing, truncation errors, widely different signal intensities, nonlinear phase distortions, and partly overlapping signals.

Investigating metal-binding in proteins by nuclear magnetic resonance

Abstract.Metal ions play a key role for the function of many proteins. The interaction of the metal ion with the protein and its involvement in the function of the protein vary widely. In some proteins, the metal ion is bound tightly to the ligand residues and may be the key player in the function of the protein, as in the case of blue copper proteins. In other proteins, the metal ion is bound only temporarily and loosely to the protein, as in the case of some metalloenzymes and other proteins where the metal ion acts as a cofactor necessary for the function of the protein. Such proteins are often known as metal ion-activated proteins. The review focuses on recent nuclear magnetic resonance (NMR) studies of a series of metal-dependent proteins and the characterization of the metal-binding sites. In particular, we focus on NMR techniques for studying metal binding to proteins such as chemical shift mapping, paramagnetic NMR and changes in backbone dynamics upon metal binding.

Probing electric fields in proteins in solution by NMR spectroscopy

Electric fields generated in native proteins affect almost every aspect of protein function. We present a method that probes changes in the electric field at specific locations within a protein. The method utilizes the dependence of the amide 1H and 15N NMR chemical shifts on electric charges in proteins. Charges were introduced at different positions in the blue copper protein plastocyanin, by protonation of side chains or by substitution of the metal ion. It is found that the associated chemical shift perturbations (CSPs) stem mainly from long‐range electric field effects caused by the change in the electric charge. It is demonstrated that the CSPs can be used to estimate the dielectric constant at different locations in the protein, estimate the nuclear shielding polarizability, or position charges in proteins. Proteins 2008.

Enhanced Stability of a Protein with Increasing Temperature

The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility of the backbone of the helices decreases with temperature in the range from 24 °C to ∼40 °C, corresponding to an increasing stability. A concomitant increase with temperature of the electrostatic interactions between charged residues forming an interhelical network of salt bridges at the center of the four-helix core suggests that these interactions give rise to the decreasing flexibility and increasing stability of the protein. However, numerous hydrophobic interactions in the interior of the four-helix core may also contribute. Above ∼40 °C, where the thermal energy overcomes the electrostatic and hydrophobic interactions, a substantial increase in the flexibility of the helix backbones results in a highly positive contribution from the local conformational heat capacity, C(p, conf), of the helix backbones to the total heat capacity, C(p), of the protein. This reduces the change in heat capacity upon unfolding, ΔC(p), increases the change in the Gibbs free energy, ΔG(unfold), and stabilizes the protein at high temperatures. A similar decrease in flexibility is found near other salt bridges in hGH and in Calmodulin and may be of general importance for the thermostability of proteins and, in particular, of the salt bridge intensive thermophilic proteins.

NMR studies of the aggregation of glucagon-like peptide-1: formation of a symmetric helical dimer

Nuclear magnetic resonance (NMR) spectroscopy reveals that higher‐order aggregates of glucagon‐like peptide‐1‐(7–36)‐amide (GLP‐1) in pure water at pH 2.5 are disrupted by 35% 2,2,2‐trifluoroethanol (TFE), and form a stable and highly symmetric helical self‐aggregate. NMR spectra show that the helical structure is identical to that formed by monomeric GLP‐1 under the same experimental conditions [Chang et al., Magn. Reson. Chem. 37 (2001) 477–483; Protein Data Bank at RCSB code: 1D0R], while amide proton exchange rates reveal a dramatic increase of the stability of the helices of the self‐aggregate. Pulsed‐field gradient NMR diffusion experiments show that the TFE‐induced helical self‐aggregate is a dimer. The experimental data and model calculations indicate that the dimer is a parallel coiled coil, with a few hydrophobic residues on the surface that may cause aggregation in pure water. The results suggest that the coiled coil dimer is an intermediate state towards the formation of higher aggregates, e.g. fibrils.

Characterization of Conformational Exchange of a Histidine Side Chain: Protonation, Rotamerization, and Tautomerization of His61 in Plastocyanin from Anabaena variabilis

A model describing conformational exchange of His 61 in plastocyanin from Anabaena variabilis is presented. A detailed picture of the exchange dynamics has been obtained using solution NMR relaxation measurements, chemical shift titrations, and structural information provided by a high-resolution crystal structure of the protein. A three-site model for chemical exchange that involves interconversion among the tautomeric and protonated forms of the histidine side chain with rates that are fast on the NMR chemical shift time scale can account for all of the data. In general, in the limit of fast exchange, it is not possible to obtain separate measures of chemical shift differences and populations of the participating states using NMR. However, we show here that when the data mentioned above are combined, it is possible to extract values of all of the parameters that characterize the exchange process, including rates, populations, and chemical shift changes, and to provide cross-validations that establish their accuracy. The methodology is generally applicable to the study of histidine side chain dynamics, which can play an important functional role in many protein systems.

Carbon dioxide hydration activity and metal—substrate distances of manganese (II) human carbonic anhydrase B determined by 13C magnetization—transfer NMR

A CO2 hydration activity for Mn(II) human carbonic anhydrase B (MnHCAB) of 7% of the activity of the native Zn2+ enzyme has been determined using a 13C magnetization—transfer NMR approach, that involves two complementary experiments. As this approach also allows a determination of the individual relaxation rates of the enzyme‐bound CO2 and HCO− 3, an evaluation could be made of the distances between these substrates and the paramagnetic Mn2+ in the active site. Thus HCO− 3 is found to bind directly to Mn2+, whereas CO2 is attached relatively weakly to the enzyme without a direct bond to the metal ion.

Two-dimensional linear-prediction NMR spectroscopy

Abstract A two-dimensional linear-prediction procedure, applying the linear-prediction principle to both dimensions, is described in detail. It is demonstrated that a two-dimensional linear-prediction calculation is feasible because the most time-consuming parts of the procedure are only performed once for a given 2D data set. Deviations from ideality caused by noise and short data records are discussed, and it is shown how to obtain maximum information in these cases. The advantages of the method as compared to the two-dimensional Fourier analysis are illustrated on a 1 H COSY free-induction decay of threonine.