Solid-state NMR methods for oriented membrane proteins.

Oriented-sample solid-state NMR represents one of few experimental methods capable of characterising the membrane-bound conformation of proteins in the cell membrane. Since the technique was developed 25 years ago, the technique has been applied to study the structure of helix bundle membrane proteins and antimicrobial peptides, characterise protein-lipid interactions, and derive information on dynamics of the membrane anchoring of membrane proteins. We will review the major developments in various aspects of oriented-sample solid-state NMR, including sample-preparation methods, pulse sequences, theory required to interpret the experiments, perspectives for and guidelines to new experiments, and a number of representative applications.

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Bicyclic peptide inhibitor of urokinase-type plasminogen activator: mode of action.

The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide-protease interactions will aid future designs of bicyclic peptide protease inhibitors.

Designing dipolar recoupling and decoupling experiments for biological solid-state NMR using interleaved continuous wave and RF pulse irradiation.

Rapid developments in solid-state NMR methodology have boosted this technique into a highly versatile tool for structural biology. The invention of increasingly advanced rf pulse sequences that take advantage of better hardware and sample preparation have played an important part in these advances. In the development of these new pulse sequences, researchers have taken advantage of analytical tools, such as average Hamiltonian theory or lately numerical methods based on optimal control theory. In this Account, we focus on the interplay between these strategies in the systematic development of simple pulse sequences that combines continuous wave (CW) irradiation with short pulses to obtain improved rf pulse, recoupling, sampling, and decoupling performance. Our initial work on this problem focused on the challenges associated with the increasing use of fully or partly deuterated proteins to obtain high-resolution, liquid-state-like solid-state NMR spectra. Here we exploit the overwhelming presence of (2)H in such samples as a source of polarization and to gain structural information. The (2)H nuclei possess dominant quadrupolar couplings which complicate even the simplest operations, such as rf pulses and polarization transfer to surrounding nuclei. Using optimal control and easy analytical adaptations, we demonstrate that a series of rotor synchronized short pulses may form the basis for essentially ideal rf pulse performance. Using similar approaches, we design (2)H to (13)C polarization transfer experiments that increase the efficiency by one order of magnitude over standard cross polarization experiments. We demonstrate how we can translate advanced optimal control waveforms into simple interleaved CW and rf pulse methods that form a new cross polarization experiment. This experiment significantly improves (1)H-(15)N and (15)N-(13)C transfers, which are key elements in the vast majority of biological solid-state NMR experiments. In addition, we demonstrate how interleaved sampling of spectra exploiting polarization from (1)H and (2)H nuclei can substantially enhance the sensitivity of such experiments. Finally, we present systematic development of (1)H decoupling methods where CW irradiation of moderate amplitude is interleaved with strong rotor-synchronized refocusing pulses. We show that these sequences remove residual cross terms between dipolar coupling and chemical shielding anisotropy more effectively and improve the spectral resolution over that observed in current state-of-the-art methods.

Rapid solid-state NMR of deuterated proteins by interleaved cross-polarization from ¹H and ²H nuclei.

We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from (1)H and (2)H spins, taking advantage of their different T(1) relaxation times. Different (1)H- and (2)H-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) (1)H→(13)C/(2)H→(13)C CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly (2)H, (15)N,(13)C-labeled α-spectrin SH3 domain sample with all or 30% back-exchanged labile (2)H to (1)H. It is demonstrated how 1D (13)C CP/MAS or 2D (13)C-(13)C correlation spectra initialized with polarization from either (1)H or (2)H may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D (13)C-(13)C correlation spectra may be recorded interleaved with (2)H-(13)C correlation spectra to obtain (13)C-(13)C correlations along with information about dynamics from (2)H sideband patterns.

Optimal (2)H rf Pulses and (2)H-(13)C Cross-Polarization Methods for Solid-State (2)H MAS NMR of Perdeuterated Proteins.

We present a novel concept for rf pulses and optimal control designed cross-polarization experiments for quadrupolar nuclei. The methods are demonstrated for (2)H CP-MAS and (2)H multiple-pulse NMR of perdeuterated proteins, for which sensitivity enhancements up to an order of magnitude are presented relative to commonly used approaches. The so-called RESPIRATION rf pulses combines the concept of short broad-band pulses with generation of pulses with large flip angles through distribution of the rf pulse over several rotor echoes. This lead to close-to-ideal rf pulses, facilitating implementation of experiments relying on the ability to realize high-performance 90 and 180° pulses, as, for example, in refocused INEPT and double-to-single quantum coherence experiments, or just pulses that provide a true representation of the quadrupolar powder pattern to extract information about the structure or dynamics. The optimal control (2)H → (13)C CP-MAS method demonstrates transfer efficiencies up to around 85% while being extremely robust toward rf inhomogeneity and resonance offsets.

Residue-specific information about the dynamics of antimicrobial peptides from (1)H-(15)N and (2)H solid-state NMR spectroscopy.

We present a new method to obtain information about the conformational dynamics of membrane-proteins using solid-state NMR experiments of oriented samples. By measuring the orientation-dependent (1)H-(15)N dipole-dipole coupling, (15)N anisotropic chemical shift, and (2)H quadrupole coupling parameters for a single residue, it is possible to obtain information about the local dynamics of each residue in the protein. This may be interpreted on an individual basis or through models extended to study conformational motion of membrane-protein segments. The method is demonstrated for the antimicrobial peptaibol alamethicin for which combined analysis of anisotropic interactions for the Aib(8) residue provides detailed information about helix-tilt angle, wobbling, and oscillatory rotation around the helix axis in the membrane bound state. This information is in very good agreement with coarse-grained MD simulations of the peptide in lipid bilayers.