Insights into Protein Dynamics from ¹⁵N-¹H HSQC

Protein dynamic information is customarily extracted from 15N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained from the 1H line widths in 15N-1H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed computer programs to predict amide proton line widths from (crystal) structures. We aim to answer the following basic questions: is the 1H linewidth of a HSQC cross peak smaller than average because its 1H nucleus has few dipolar neighbors, or because the resonance is motionally narrowed? Is a broad line broad because of conformational exchange, or because the 1H nucleus resides in a dense proton environment? We calibrate our programs by comparing computational and experimental results for GB1 (58 residues). We deduce that GB1 has low average 1HN order parameters (0.8), in broad agreement with what was found by others from 15N relaxation experiments (Idiyatullin et al., 2003). We apply the program to the BPTI crystal structure and compare the results with a 15N-1H HSQC spectrum of BPTI (56 residues) and identify a cluster of conformationally broadened 1HN resonances that belong to an area, for which millisecond dynamics has been previously reported from 15N relaxation data (Szyperski et al., J. Biomol. NMR 3, 151-164, 1993). We feel that our computational approach is useful to glean insights into the dynamical properties of larger biomolecules for which high-quality 15N relaxation data cannot be recorded.

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand–protein interaction.

Bacterial Cell Wall Analogue Peptides Control the Oligomeric States and Activity of the Glycopeptide Antibiotic Eremomycin: Solution NMR and Antimicrobial Studies

For some time, glycopeptide antibiotics have been considered the last line of defense against Methicillin-resistant Staphylococcus aureus (MRSA). However, vancomycin resistance of Gram-positive bacteria is an increasingly emerging worldwide health problem. The mode of action of glycopeptide antibiotics is essentially the binding of peptidoglycan cell-wall fragments terminating in the d-Ala-d-Ala sequence to the carboxylate anion binding pocket of the antibiotic. Dimerization of these antibiotics in aqueous solution was shown to persist and even to enhance the antibacterial effect in a co-operative manner. Some works based on solid state (ss) Nuclear Magnetic Resonance (NMR) studies questioned the presence of dimers under the conditions of ssNMR while in a few cases, higher-order oligomers associated with contiguous back-to-back and face-to-face dimers were observed in the crystal phase. However, it is not proved if such oligomers persist in aqueous solutions. With the aid of 15N-labelled eremomycin using 15N relaxation and diffusion NMR methods, we observed tetramers and octamers when the N-Ac-d-Ala-d-Ala dipeptide was added. To the contrary, the N-Ac-d-Ala or (N-Ac)2-l-Lys-d-Ala-d-Ala tripeptide did not induce higher-order oligomers. These observations are interesting examples of tailored supramolecular self-organization. New antimicrobial tests have also been carried out with these self-assemblies against MRSA and VRE (resistant) strains.

Protein dynamics insights from ¹⁵N-¹H (TROSY) HSQC

Protein dynamic information is customarily extracted from 15N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained form the 1H line widths in 15N-1H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed a computer program to predict amide proton line widths from (crystal) structures. As a calibration, we test our approach on BPTI. We find that we can predict most of the distribution of experimental amide proton line widths if we take the dipole-dipole interaction with at least 40 surrounding protons into account. When focusing our attention the outliers of the distribution, we find for BPTI a cluster of conformationally broadened 1HN resonances of residues in strands 10–15 and 36–40 of the beta sheet. Conformational exchange broadening of the 15NH resonances for these residues was previously reported using 15N relaxation measurements (Szyperski et al., J. Biomol. NMR 3, 151–164, 1993). There is little or no evidence for motional narrowing of the 1HN resonances, also in agreement with earlier data using 15N relaxation methods (Beeser et.al, J. Mol. Biol. 269, 154–164, 1997). We also apply our program to 42 kDa domain of the human Hsc70 protein. In this case, there is no previous 15N relaxation data to compare with, but we find, again from the outliers of the distribution, both exchange broadening and motional narrowing that appears to corroborate previous conformational insights for this domain.

Dynamics-Derived Insights into Complex Formation between the CXCL8 Monomer and CXCR1 N-Terminal Domain: An NMR Study

Interleukin-8 (CXCL8), a potent neutrophil-activating chemokine, exerts its function by activating the CXCR1 receptor that belongs to class A G protein-coupled receptors (GPCRs). Receptor activation involves interactions between the CXCL8 N-terminal loop and CXCR1 N-terminal domain (N-domain) residues (Site-I) and between the CXCL8 N-terminal and CXCR1 extracellular/transmembrane residues (Site-II). CXCL8 exists in equilibrium between monomers and dimers, and it is known that the monomer binds CXCR1 with much higher affinity and that Site-I interactions are largely responsible for the differences in monomer vs. dimer affinity. Here, using backbone 15N-relaxation nuclear magnetic resonance (NMR) data, we characterized the dynamic properties of the CXCL8 monomer and the CXCR1 N-domain in the free and bound states. The main chain of CXCL8 appears largely rigid on the picosecond time scale as evident from high order parameters (S2). However, on average, S2 are higher in the bound state. Interestingly, several residues show millisecond-microsecond (ms-μs) dynamics only in the bound state. The CXCR1 N-domain is unstructured in the free state but structured with significant dynamics in the bound state. Isothermal titration calorimetry (ITC) data indicate that both enthalpic and entropic factors contribute to affinity, suggesting that increased slow dynamics in the bound state contribute to affinity. In sum, our data indicate a critical and complex role for dynamics in driving CXCL8 monomer-CXCR1 Site-I interactions.