Three-Dimensional Triple-Resonance NMR of \u3csup\u3e13\u3c/sup\u3eC/\u3csup\u3e15\u3c/sup\u3eN-Enriched Proteins Using Constant-Time Evolution

Recently it has been convincingly demonstrated that 30 triple-resonance NMR provides a practical alternative for obtaining sequential resonance assignments in larger proteins ( 1, 2). This approach requires a set of five or six 30 NMR experiments that correlate the various protein backbone nuclei. Details regarding the mechanisms and technical implementations of these experiments have been described previously ( 3- 5). Two of the experiments used in this approach correlate backbone Hα and Cα resonances with either the intraresidue carbonyl resonance (CO) or the 15N resonance of the succeeding residue and are referred to as HCACO and HCA(CO)N, respectively. The present Communication describes a modification of these experiments which optimizes their sensitivity and removes the F1 antiphase character of correlations

Relationships between the Precision of High-Resolution Protein NMR Structures, Solution-Order Parameters, and Crystallographic B Factors

One of the principal motivations for studying proteins by nuclear magnetic resonance stems from the desire to describe the solution structure of these molecules as compared to the generally perceived static picture obtained by X-ray crystallography. Indeed, it is one of the unique features of NMR spectroscopy that in addition to structural data, dynamic properties can be probed and characterized by measuring relaxation parameters. Furthermore, any mobility of the protein in solution will necessarily modulate the measured NMR parameters and should influence the resulting structure. It has been argued that regions of a protein that are highly mobile would be expected to be defined to a lesser degree of precision than regions that are rigid (1. 2 )

Encounter complexes and dimensionality reduction in protein-protein association

An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition

Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex

AbstractThe solution structure of the specific complex between the high mobility group (HMG) domain of SRY (hSRY-HMG), the protein encoded by the human testis-determining, gene, and its DNA target site in the promoter of the Müllerian inhibitory substance gene has been determined by multidimensional NMR spectroscopy. hSRY-HMG has a twisted L shape that presents a concave surface (made up of three helices and the N- and C-terminal strands) to the DNA for sequence-specific recognition. Binding of hSRY-HMG to its specific target site occurs exclusively in the minor groove and induces a large conformational change in the DNA. The DNA in the complex has an overall 70°–80° bend and is helically unwound relative to classical A- and B-DNA. The structure of the complex reveals the origin of sequence-specific binding within the HMG-1/HMG-2 family and provides a framework for understanding the effects of point mutations that cause 46X,Y sex reversal at the atomic level

The solution structure of human thioredoxin complexed with its target from Ref-1 reveals peptide chain reversal

AbstractBackground: Human thioredoxin (hTRX) is a 12 kDa cellular redox protein that has been shown to play an important role in the activation of a number of transcriptional and translational regulators via a thiol-redox mechanism. This activity may be direct or indirect via another redox protein known as Ref-1. The structure of a complex of hTRX with a peptide comprising its target from the transcription factor NFκB has previously been solved. To further extend our knowledge of the recognition by and interaction of hTRX with its various targets, we have studied a complex between hTRX and a Ref-1 peptide. This complex represents a kinetically stable mixed disulfide intermediate along the reaction pathway.Results Using multidimensional heteronuclear edited and filtered NMR spectroscopy, we have solved the solution structure of a complex between hTRX and a 13-residue peptide comprising residues 59–71 of Ref-1. The Ref-1 peptide is located in a crescent-shaped groove on the surface of hTRX, the groove being formed by residues in the active-site loop (residues 32–36), helix 3, β strands 3 and 5, and the loop between β strands 3 and 4. The complex is stabilized by numerous hydrogen-bonding and hydrophobic interactions that involve residues 61–69 of the peptide and confer substrate specificity.Conclusion The orientation of the Ref-1 peptide in the hTRX–Ref-1 complex is opposite to that found in the previously solved complex of hTRX with the target peptide from the transcription factor NFκB. Orientation is determined by three discriminating interactions involving the nature of the residues at the P−2, P−4 and P−5 binding positions. (P0 defines the active cysteine of the peptide, Cys65 for Ref-1 and Cys62 for NFκB. Positive and negative numbers indicate residues N-terminal and C-terminal to this residue, respectively, and vice versa for NFκB as it binds in the opposite orientation.) The environment surrounding the reactive Cys32 of hTRX, as well as the packing of the P+3 to P−4 residues are essentially the same in the two complexes, despite the opposing orientation of the peptide chains. This versatility in substrate recognition permits hTRX to act as a wide-ranging redox regulator for the cell

Estimation of interdomain flexibility of N-terminus of factor H using residual dipolar couplings

Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e. FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC datasets, collected in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC datasets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of < 40 °. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3), could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex