Mark S. Friedrichs

Characterization of the three-dimensional solution structure of human profilin: proton, carbon-13, and nitrogen-15 NMR assignments and global folding pattern

Abstract Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.

A model-free algorithm for the removal of baseline artifacts.

SummaryA novel algorithm for removing baseline distortions in NMR spectra is presented. The algorithm approximates the baseline as the median of the noise extrema. Consequently, the method does not require that NMR peaks be discriminated from noise peaks. In addition, no assumptions regarding the source or functional form of the distortion are made. The algorithm is shown to remove the baseline artifacts present in a particularly distorted NOESY spectrum and to reveal peaks which had been obscured by the artifacts. The parameters and spectral characteristics (signal-to-noise ratio, NMR peak density, peak linewidths) governing the resolution of the calculated baselines are also explored.

An automated procedure for the assignment of protein 1HN, 15N, 13Cα, 1Hα, 13Cβ and 1Hβ resonances

SummaryA computer algorithm that determines the 1HN, 15N, 13Cα, 1Hα, 13Cβ chemical-shift assignments of protein residues with minimal human intervention is described. The algorithm is implemented as a suite of macros that run under a modified version of the FELIX 1.0 program (Hare Research, Bothell, WA). The input to the algorithm is obtained from six multidimensional, triple-resonance experiments: 3D HNCACB, 3D CBCA(CO)HN, 4D HNCAHA, 4D HN(CO)CAHA, 3D HBHA(CO)NH and 3D HNHA(Gly). For small proteins, the two 4D spectra can be replaced by either the 3D HN(CA)HA, 3D H(CA)NNH, or the 15N-edited TOCSY-HSQC experiments. The algorithm begins by identifying and collecting the intraresidue and sequential resonances of the backbone and 13Cβ atoms into groups. These groups are sequentially linked and then assigned to residues by matching the 13Cα and 13Cβ chemical-shift profiles of the linked groups to that of the proteins primary structure. A major strength of the algorithm is its ability to overcome imperfect data, e.g., missing or overlapping peaks. The viability of the procedure is demonstrated with two test cases. In the first, NMR data from the six experiments listed above were used to reassign the backbone resonances of the 93-residue human hnRNP C RNA-binding domain. In the second, a simulated cross-peak list, generated from the published NMR assignments of calmodulin, was used to test the ability of the algorithm to assign the backbone resonances of proteins containing internally homologous segments. Finally, the automated method was used to assign the backbone resonances of apokedarcidin, a previously unassigned, 114-residue protein.

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

SummaryThe 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys1 with Cys13 and Cys7 with Cys19, and the side chain of Asp9 forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H2O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.

Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy

SummaryExtensive 1H and 13C assignments have been obtained for the aliphatic resonances of a uniformly 13C-and 15N-labeled recombinant VL domain from the anti-digoxin antibody 26-10. Four-dimensional triple resonance NMR data acquired with the HNCAHA and HN(CO)CAHA pulse sequences [Kay et al. (1992) J. Magn. Reson., 98, 443–450] afforded assignments for the backbone HN, N, Hα and Cα resonances. These data confirm and extend HN, N and Hα assignments derived previously from three-dimensional 1H-15N NMR studies of uniformly 15N-labeled VL domain [Constantine et al. (1992), Biochemistry, 31, 5033–5043]. The identified Hα and Cα resonances provided a starting point for assigning the side-chain aliphatic 1H and 13C resonances using three-dimensional HCCH-COSY and HCCH-TOCSY experiments [Clore et al. (1990), Biochemistry, 29, 8172–8184]. The Cα and Cβ chemical shifts are correlated with the VL domain secondary structure. The extensive set of side-chain assignments obtained will allow a detailed comparison to be made between the solution structure of the isolated VL domain and the X-ray structure of the VL domain within the 26–10 Fab.

Relaxation study of the backbone dynamics of human profilin by two‐dimensional 1H‐15N NMR

The dynamic properties of 111 backbone HN sites in uncomplexed human profilin, a protein of 139 residues, have been characterized by two‐dimensional inverse‐detected 1H‐15N NMR spectroscopy. Heteronuclear {1H}‐15N nuclear Overhauser effects and 15N longitudinal and transverse relaxation rates have been analyzed in terms of model‐free spectral density functions and exchange contributions to transverse relaxation rates. Relatively high mobilities on the nanosecond timescale are observed for Asp26 and Ser27, which form part of a loop connecting β‐strands A and B, and for Thr92 through Ala95, which are in a loop connecting β‐strands E and F. Significant exchange contributions, indicative of motions on the microsecond to millisecond timescale, have been obtained for 30 residues. These include Leu77, Asp80 and Gly81 of a loop between β‐strands D and E, Ser84 and Met85 of β‐strand E, Gly121 of a loop connecting β‐strand G and the C‐terminal helix, and Gln138, which is next to the C‐terminal residue Tyr139. Some of the regions showing high flexibility in profilin are known to be involved in poly‐L‐proline binding.