Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal-mol'1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.The ordered, functional structures of proteins reflect two tendencies that are often opposed. On one hand, proteins fold to minimize their free energy. On the other hand, they organize themselves to recognize a ligand or a transition state (1). Minimizing free energy leads to well-packed hydrophobic interiors and hydrophilic exteriors (2). Maximizing function leads to active-site clefts where charged and polar groups are sequestered from water (3, 4) and where hydrophobic patches are exposed to solvent.The hypothesis that there is a balance between stability and function can be stated most strongly as follows: protein residues that contribute to catalysis or ligand binding are not optimal for protein stability. This "stability-function" hypothesis predicts that it usually should be possible to replace residues known to be important for function, reducing protein activity but concomitantly increasing stability of the folded protein.Here we describe experiments that directly test the stabilityfunction hypothesis in T4 lysozyme, an enzyme well characterized for the effects of mutation on structure and stability (5). Five residues were replaced (Table 1 and Fig. 1). These included two residues implicated in chemical catalysis, 11,12), as well as thyee others thought to have a role in substrate binding, Gly-30, Ser-117, and Asp-132. We measured the thermodynamic stability and kinetic activity of the mutant lysozymes. To determine the structural consequences of the substitutions, we determined x-ray crystal structures for several of these proteins. MATERIALS AND METHODSMutagenesis and Protein Purification. Mutations were introduced into the T4 lysozyme gene borne by M13 phage derivative M13mpl8 T4e by mismatched oligonucleotides using the method of Kunkel et al (13) as detailed (6, 14).

Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is caused by the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified Ϸ87% (819/942) of the hydrogen positions in PYP and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs and the mechanism of the formation of LBHBs.neutron crystallography ͉ photoreaction ͉ proton translocation ͉ short hydrogen bond T he idea that the formation of low-barrier hydrogen bonds (LBHBs) plays an essential role in enzyme catalysis was proposed in the early 1990s (1, 2). Although several lines of circumstantial evidence support the existence of LBHBs, negative results have also been published (3-5). This discrepancy is caused by the absence of direct demonstration of LBHBs in proteins. In general, hydrogen bonds in proteins are identified by the distance between a donor and an acceptor within the crystal structure. Because of its abnormally short bond length, a LBHB is accompanied by a quasi-covalent bond feature, whereas an ordinary hydrogen bond can be depicted as an electrostatic interaction between a donor-proton dipole and a dipole (or a monopole) on an acceptor atom (6-8). In LBHBs, the proton is shared by the donor and acceptor atoms, resulting in the distribution of the hydrogen between the two (6). Therefore, to identify a LBHB, it is essential to determine the position of the hydrogen atom and those of the donor and acceptor atoms. Recently, it was shown that a light sensor protein, photoactive yellow protein (PYP), contains 2 short hydrogen bonds (SHBs) adjacent to the reaction center, even in the ground state (9, 10). The hydrogen atoms involved in the SHBs, however, could not be observed either by X-ray crystallography at atomic resolution (9, 11) or neutron crystallography at 2.5-Å resolution (10).PYP is a putative photoreceptor for negative phototaxis of the purple phototropic bacterium, Halorhodospira halophila (12). The chromophore of PYP, p-coumaric acid (pCA), is buried in a hydrophobic pocket. Absorption of a photon triggers the isomerization of the chromophore and the subsequent thermal reaction cycle (13,14). The hydrogen-bonding network near the chromophore is modulated during the thermal reaction, result...

The crystal structure of poly(l-lactic acid) (PLLA) α form has been analyzed in detail by utilizing the 2-dimensional wide-angle X-ray (WAXD) and neutron diffraction (WAND) data measured for the ultradrawn sample. The WAXD data were collected using a synchrotron-sourced high-energy X-ray beam of wavelength 0.328 Å at SPring-8, Japan and the WAND data were measured using a neutron beam of wavelength 1.510 Å with a cylindrical imaging-plate camera of BIX-3 system at Japan Atomic Energy Agency. The initial crystal structure model was extracted successfully by a direct method under the assumption of the space group P212121 using about 700 X-ray reflections observed at −150 °C, the number of which was overwhelmingly large compared with the data reported by the previous other researchers and allowed us to perform more precise structural analysis. The crystal structure model obtained by the direct method was refined so that the best agreement between the observed and calculated integrated intensities was obtained or the reliability factor (R) became minimal: R was 18.2% at −150 °C and 23.2% at 25 °C. The thus-obtained chain conformation took the distorted (10/3) helical form with 21 helical symmetry along the chain axis. However, the symmetrically forbidden reflections 003, 007, 009 etc. were detected in a series of the 00L reflections, requiring us to erase the 21 screw symmetry along the molecular chain. By assuming the space group symmetry P1211, the structural refinement was made furthermore and the finally obtained R factor was 19.3% at −150 °C and 19.4% at 25 °C. Although the structural deviation from the 21 screw symmetry was only slightly, this refined model was found to reproduce the observed reflection profiles well for all the layer lines. The thus X-ray-analyzed crystal structure was transferred to the WAND data analysis to determine the hydrogen atomic positions. The R factor was 23.0% for the 92 observed reflections at 25 °C. The agreement between the observed and calculated layer line profiles was good. In this way the crystal structure of PLLA α form has been established on the basis of both the X-ray and neutron diffraction analyses.

The glycosyl-enzyme intermediate in lysozyme action has long been considered to be an oxocarbonium ion, although precedent from other glycosidases and theoretical considerations suggest it should be a covalent enzyme-substrate adduct. The mutation of threonine 26 to glutamic acid in the active site cleft of phage T4 lysozyme (T4L) produced an enzyme that cleaved the cell wall of Escherichia coli but left the product covalently bound to the enzyme. The crystalline complex was nonisomorphous with wild-type T4L, and analysis of its structure showed a covalent linkage between the product and the newly introduced glutamic acid 26. The covalently linked sugar ring was substantially distorted, suggesting that distortion of the substrate toward the transition state is important for catalysis, as originally proposed by Phillips. It is also postulated that the adduct formed by the mutant is an intermediate, consistent with a double displacement mechanism of action in which the glycosidic linkage is cleaved with retention of configuration as originally proposed by Koshland. The peptide part of the cell wall fragment displays extensive hydrogen-bonding interactions with the carboxyl-terminal domain of the enzyme, consistent with previous studies of mutations in T4L.