A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.
Previous rotamer libraries showed little significant clustering for asparagine 2 or glutamine 3 values, but none of those studies corrected amide orientations or omitted disordered side chains. The current survey used 240 proteins at <1.7 Å resolution with <50% homology and <30 clashes per thousand atoms (atomic overlap >0.4 Å). All H atoms were added and optimized, and amide orientation was f lipped by 180°if required by H bonding or atomic clashes. A side chain was included only if its amide orientation was clearly determined and if no atom had a B factor >40, alternate conformation, or severe clash; that selection process yielded 1,490 Asn and 863 Gln side chains. Clear clustering was observed for Asn 2 and Gln 3 (except when Gln 2 is trans). For Gln, five major and four minor rotamers cover 87% of examples. For Asn, there are seven backbone-independent rotamers covering 94% of examples plus rotamers specified for strictly ␣-helical, , and left-handed (؉) Asn. Although the strongest inf luence on angles is avoidance of atomic clashes (especially with the NH 2 hydrogens), some Asn or Gln rotamers are inf luenced by favorable van der Waals contacts and others by specific local H-bond patterns.The most important variables for protein conformation are the , , and angles of the backbone, which describe overall tertiary structure. However, the side-chain 1 , 2 , 3 . . . dihedral angles constitute the other half of the conformational specification, determining how the parts of the protein fit together and how the functional groups of the side chains can interact with other molecules. Originally, each -angle distribution was studied separately, but since Ponder and Richards (1), most treatments have been organized around ''rotamers,'' or separate populated clusters in the n-dimensional space of values for a given amino acid type. Libraries of discrete side chain rotamers are widely used for homology modeling, protein redesign, Monte Carlo calculations, and crystallographic electron-density map fitting. The usefulness of the rotamer concept depends on two conditions: (i) the clusters are sharp and separated and (ii) the distributions are not simply independent of one another. Both conditions hold for most side-chain types, but Asn and Gln show notably poor clustering.Asn and Gln 1 and Gln 2 angles are well behaved, with optima near the three staggered values. The major aspect at issue in the analysis of Asn͞Gln conformations is the outermost dihedral angle ( 2 for Asn and 3 for Gln). When the side-chain amide N and O atoms cannot be distinguished, that angle is uncertain by 180°. It involves the rotation of a planar amide relative to a tetrahedral group and has less distinct preferences than a rotation between methyl or methylene groups. The Asn͞Gln amide angles are often omitted from side-chain conformer analyses and are treated differently each time they are included. Different studies have presumed 180°s ymmetry (2-4); have used 0°, ϩ, t, and Ϫ classes (1); have used Ͻ180°and Ͼ180°classes (5); have used bi...
When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all-atom contacts~including hydrogens! to answer that question. The interactive MAGE0PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all-atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active-site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild-type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.
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