Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme frombacteriopbage T4 were used to explore the feasibility of usingsite-directed mutagenesis to generate isomorphous heavy-atomderivatives for protein crystallography. Cysteines 54 and 97,present in wild-type lysozyme, can be readily reacted with mercuricion to produce an excellent isomorphous heavy-atom derivative.Mutants with an additional cysteine at position 86,146,153 or157, or with Cys 97 replaced by Val, were engineered by site-directedmutagenesis. The mutant lysozyme Thr 157 - Cys reacts with mercuricchloride to give an excellent new derivatve although Cys 157is only -60% substituted with the heavy atom. The cysteine atposition 146 is largely buried but reacts readily with mercuricchloride. In this case the isomorphism is poor and the resultantderivative is of marginal quality. Cys 153 reacts rapidly withmercuric ion but the derivative crystals do not diffract. Themutant Pro 86 - Cys does not yield a particularly good heavy-atomderivative. This is due in part to a loss of isomorphism associatedwith the mutation. In addition, Cys 86 shows very little reactivitytowards mercurials even though it is fully exposed to solvent.The mutation Cys 97 Val was used to explore the possibilityof creating an independent derivative by deleting a heavy-atomsite already present in wild-type lysozyme. In all cases thatwere tested, the quality of the heavy-atom derivative was improvedby using as an isomorphous pair mercury-substituted mutant versusnon-substituted mutant rather than mercury-substituted mutantversus (non-substituted) wild-type lysozyme. Unexpectedly, thecysteines that are most exposed to solvent and most mobile areleast reactive toward mercuric chloride. The cysteines thatprovide the best heavy-atom sites are those that are locatedin surface crevices and are only partly exposed to solvent.     

A molecular dynamics simulation of bacteriophage T4 lysozyme

An analysis of a 400 ps molecular dynamics simulation of the164 amino acid enzyme T4 lysozyme is presented. The simulationwas carried out with all hydrogen atoms modeled explicitly,the inclusion of all 152 crystallographic waters and at a temperatureof 300 K. Temporal analysis of the trajectory versus energy,hydrogen bond stability, r.m.s. deviation from the startingcrystal structure and radius of gyration, demonstrates thatthe simulation was both stable and representative of the averageexperimental structure. Average structural properties were calculatedfrom the enzyme trajectory and compared with the crystal structure.The mean value of the C displacements of the average simulatedstructure from the X-ray structure was 1.1 ± 0.1 Å;differences of the backbone and angles between the averagesimulated structure and the crystal structure were also examined.Thermal-B factors were calculated from the simulation for heavyand backbone atoms and both were in good agreement with experimentalvalues. Relationships between protein secondary structure elementsand internal motions were studied by examining the positionalfluctuations of individual helix, sheet and turn structures.The structural integrity in the secondary structure units waspreserved throughout the simulation; however, the A helix didshow some unusually high atomic fluctuations. The largest backboneatom r.m.s. fluctuations were found in non-secondary structureregions; similar results were observed for r.m.s. fluctuationsof non-secondary structure and angles. In general, the calculatedvalues of r.m.s. fluctuations were quite small for the secondarystructure elements. In contrast, surface loops and turns exhibitedmuch larger values, being able to sample larger regions of conformationalspace. The C difference distance matrix and super-positioninganalyses comparing the X-ray structure with the average dynamicsstructure suggest that a ‘hinge-bending’ motionoccurs between the N- and C-terminal domains.     

Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS

KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.     

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate     

Theoretical studies on solvation contribution to the thermodynamic stability of mutants of lysozyme T4

Atomic solvation parameters (ASPs) are widely used to estimatethe solvation contribution to the thermodynamic stability ofproteins as well as the free energy of association for protein–ligandcomplexes. In view of discrepancies in the results of free energiesof solvation of folding for various proteins obtained usingdifferent atomic solvation parameter sets, systematic studieshave been carried out for the calculation of accessible surfacearea and the changes in free energy of solvation of folding(     

Modeling protein stability: a theoretical analysis of the stability of T4 lysozyme mutants

Free energy calculations were conducted to determine the relative stability of the unnatural amino acid mutants of T4 lysozyme norvaline (Nvl) and O-methyl-serine (Mse) and of alanine at residue 133, which is leucine in the native sequence. These calculations were performed both to assess the validity of the methodology and to gain a better understanding of the forces which contribute to protein stability. Peptides of different length were used to model the denatured state. Restraints were employed to force sampling of the side chain chi1 dihedral of the perturbed side chain, and the effect of protein repacking in response to mutation was studied through the use of different constraint sets. In addition, the convergence behavior and hysteresis of the simulations in the folded and unfolded states were determined. The calculated results agree well with experiment, + 1.84 versus + 1.56 kcal/mol for Mse-->Nvl and -3.48 versus -2.2 to -3.6 kcal/mol for Nvl-->Ala. We find that free energy calculations can provide useful insights to protein stability when conducted carefully on a well chosen system. Our results suggest that loss of packing interactions in the native state is a major source of destabilization for mutants which decrease the amount of buried nonpolar surface area and that subtle responses of the backbone affect the magnitude of the loss of stability. We show that the conformational freedom of the chi1 dihedral has a noticeable effect on protein stability and that the solvation of amino acid side chains is strongly influenced by interactions with the peptide backbone.      

The role of cysteine oxidation in the thermal inactivation of T4 lysozyme

Wild-type T4 lysozyme contains unpaired cysteine residues atpositions 54 and 97. To investigate the role these residuesplay in the thermal inactivation of the wild-type, we constructeda double mutant with these cysteines replaced with valine andserine. This molecule, T4 lysozyme (C54V/C97S), is more stablethan the wild-type to inactivation at 70°C at pH 6.5 and8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGEon the inactivated products show that the wild-type is susceptibleto varying degrees of oxidative damage, depending on bufferconditions, while the cysteine-minus mutant inactivates onlyby other pathways. The products of thermal, oxidative inactivationof the wild-type are disulfide-linked oligomers. The dependenceof inactivation rate on temperature suggests that the formationof these aggregates depends on prior thermal unfolding of theT4 lysozyme molecule.     

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesizedand cloned by ligation of 26 deoxyribooligo-nucleotide fragmentsin two steps with a linearized plasmid followed by transformation.On selection by colony hybridization and DNA sequence analysis,clone pTLY.10 was identified to contain a complete T4 lysozymesynthetic DNA. On expression under lac-promoter, unfused T4lysozyme was obtained in {small tilde}4–6% yield. Thedesign and synthesis of two putative folding mutants, flexible(Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75,were based on hierarchical principles. Both mutants lost enzymaticactivity of the wildtype. These results are readily understandableif the hierarchical organization of the structure is taken intoaccount. A possible explanation is that the catalytic sitesare blocked in both mutants.     

Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive

A number of mutations have been shown previously to stabilizeT4 lysozyme. By combining up to seven such mutations in thesame protein, the melting temperature was incrementally increasedby up to 83°C at pH 5.4 (G = 3.6 kcal/mol). This shows thatit is possible to engineer a protein of enhanced thermostabilityby combining a series of rationally designed point mutations.It is also shown that this stabilization is achieved with onlyminor, localized changes in the structure of the protein. Thisis consistent with the observation that the change in stabilityof each of the multiple mutants is, in each case, additive,i.e. equal to the sum of the stability changes associated withthe constituent single mutants. One of the seven substitutions,Asn116 Asp, changes a residue that participates in substratebinding; not surprisingly, it causes a significant loss in activity.Ignoring this mutation, there is a gradual reduction in activityas successively more mutations are combined.     

Rational design of a chimeric endonuclease targeted to NotI recognition site

A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100-150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.     

Computational design of an endo-1,4-beta-xylanase ligand binding site

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-β-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 β-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the β-xylanase proteins.     

Use of the attainable region analysis to optimize particle breakage in a ball mill

Particle size reduction is one of the most widely encountered, yet least energy efficient, processes. Therefore, potentially significant energy and cost savings exist with even the slightest increase in milling efficiency. Often one would like to mill particles to a certain size, and no smaller, while minimizing energy use and milling time. We use the attainable region (AR) analysis to optimize the comminution of silica sand particles in a bench top laboratory ball mill. When the mill is loaded with a large number of grinding media (J=volume of media/mill volume=10.7%), the breakage profiles are indistinguishable over all rotation rates investigated. However, operation at lower grinding media fill level (J=1.5%) reveals separation between the grinding profiles for different rotation rates, suggesting more efficient breakage occurs at a lower grinding media fill level for a given rotation rate. Our results show that operation at multiple speeds, fast at first and then slower (φ_c=0.03), takes advantage of the initially overlapping grinding profiles and produces a similar particle size distribution with a decreased amount of processing time—less than half the time required for the single rotation rate milling. A natural extension of this idea is continuous milling, where the first mill can operate at a higher energy input for a shorter amount of time and the second mill can operate at a lower energy input for a longer amount of time.     

均匀设计在涂料设计中的应用

介绍了均匀设计与均匀设计表的特点和使用方法,并且详细地介绍了如何运用均匀设计表进行涂料配方设计,可对涂料配方设计人员在筛选因素或收缩试验范围进行逐步择优的多因素、多水平的试验时起到有效的指导。     

Use of experimental design in development of a catalyst system

NO_x storage and reduction experiments have been performed with stationary operation of a heavy-duty diesel engine rig. An optimization of the NO_x reduction performance has been done using experimental design. The adjustable parameters in this study were cycle time, injection time, injection rate and bypass time (period of reduced flow through catalysts). NO_x was reduced by 50–60% (3.3–4.1 g/kWh) with a fuel penalty below 5%. It was shown that experimental design was efficient for optimizing the NO_x reduction and this systematic approach enabled important conclusions to be drawn about the system performance.     

Use of Antoine Equation to Relate T to Y-X Equilibrium Composition

Abstract

The current practice followed in drawing isobaric y-x phase diagrams for a binary mixture (say Components 1 and 2) is based on data for the vapor pressure of the components as a function of temperature, T.     

Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D,in which Ala92 was substituted by Asp, contains a partial Ca²⁺-bindingsite and mutant M4, in which Ala83, Gm86, Asn88 and Ala92 werereplaced by Lys, Asp, Asp and Asp respectively, contains thecomplete Ca -binding site of bovine a-lactalbumin. The Ca²⁺-bindingconstants of wild type human lysozyme and of mutants A92D andM4, measured at 25C and pH 7.5, were 2(1) x 102 M"1, 8(2)x l^M"1 and 9(0.5) x 10* M"1 respectively. Information gatheredfrom mkrocalorimetrk and CD spectro-scopic measurements indicatesthat the conformational changes of the M4 mutant lysozyme, inducedby Ca²⁺ binding, are smaller than those observed for bovinea-lactalbumin and for the Ca²⁺-binding equine lysozyme. At pH4.5, the thermostability of both the apo and Ca²⁺ forms of theA92D human was decreased in comparison with that of native humanlysozyme. In particular, within the apo form of this mutantan a-helix-containing sequence was destabilized. In contrast,at the same pH the thermostability of the apo and Ca²⁺ formsof the M4 mutant lysozyme was increased. The e-ammonium groupof the Lys83 side chain is assumed to be responsible for thestabilization of the apo form of this mutant.     

Use of a quantitative structure-property relationship to design larger model proteins that fold rapidly

A quantitative structure–property relationship (QSPR)was used to design model protein sequences that fold repeatedlyand relatively rapidly to stable target structures. The specificmodel was a 125-residue heteropolymer chain subject to MonteCarlo dynamics on a simple cubic lattice. The QSPR was derivedfrom an analysis of a database of 200 sequences by a statisticalmethod that uses a genetic algorithm to select the sequenceattributes that are most important for folding and a neuralnetwork to determine the corresponding functional dependenceof folding ability on the chosen attributes. The QSPR dependson the number of anti-parallel sheet contacts, the energy gapbetween the native state and quasi-continuous part of the spectrumand the total energy of the contacts between surface residues.Two Monte Carlo procedures were used in series to optimize boththe target structures and the sequences. We generated 20 fullyoptimized sequences and 60 partially optimized control sequencesand tested each for its ability to fold in dynamic MC simulations.Although sequences in which either the number of anti-parallelsheet contacts or the energy of the surface residues is non-optimalare capable of folding almost as well as fully optimized ones,sequences in which only the energy gap is optimized fold markedlymore slowly. Implications of the results for the design of proteinsare discussed.     

Use of experimental design in detergency tests with a natural soil

A balanced incomplete block design is used to obtain detergency data in a test where cloth swatches are soiled by rubbing against the skin. The design provides increased precision in the data by adjusting for differences among soilers. The wash treatments are part of a second order rotatable design in three variables: the ratio of sodium nitrilotriacetate to sodium tripolyphosphate builder, pH of the wash solution, and temperature. The effect of builder ratio was not highly significant. Soil removal increased with higher pH and went through a maximum with increase in wash temperature. Redeposition was also measured by reflectance values obtained for unsoiled areas of the swatches. Redeposition increased with increase in wash temperature. One of five papers presented at the Symposium, “Basic Aspects of Detergency,” AOCS-ISF World Congress, Chicago, September 1970.     

A systematic approach to the synthesis and design of flexible site utility systems

The paper presents a systematic approach for the synthesis of flexible utility systems satisfying varying energy demands. The approach combines benefits of total site analysis, thermodynamic analysis and mathematical optimisation. A thermodynamic efficiency curve (TEC) is developed, which gives an overview of the maximum thermodynamic efficiencies of all possible design alternatives. TEC and hardware composites guide the selection of candidate structures in the superstructure, excluding uneconomic options from the synthesis model. The integration of thermodynamics yields significant reduction in the synthesis model, addresses the impact of variable loads on the unit efficiencies, and enables a compact formulation of the design problem over long horizons of operation. The optimisation is formulated as a multi-period MILP problem that relies on new target models to describe the performance of steam turbines, condensing turbines, gas turbines and boilers. Target models account for the variation of efficiency with unit size, load and operating conditions in a simple, yet accurate way. As a result, these models are capable of accounting for the efficiency trends of realistic units.