Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy.

Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR.

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.

New developments in isotope labeling strategies for protein solution NMR spectroscopy.

The development of novel isotope labeling strategies for proteins has facilitated the study of the structure and dynamics of these molecules. In addition, the recent emergence of alternative methods of bacterial expression for obtaining isotopically labeled proteins permits the study of new classes of important proteins by solution NMR methods.

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin.

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.

A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.

A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

An HNCO-based Pulse Scheme for the Measurement of 13Cα-1Hα One-bond Dipolar couplings in 15N, 13C Labeled Proteins.

A triple resonance pulse scheme is presented for recording 13Cα-1Hα one-bond dipolar couplings in 15N, 13C labeled proteins. HNCO correlation maps are generated where the carbonyl chemical shift is modulated by the 13Cα-1Hα coupling, with the two doublet components separated into individual data sets. The experiment makes use of recently described methodology whereby the protein of interest is dissolved in a dilute solution of bicelles which orient above a critical temperature, thus permitting measurement of significant couplings (Tjandra and Bax, 1997a). An application to the protein ubiquitin is described.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.