Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-A resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.     

Probing protein hydration and conformational states in solution

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase

Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.     

Does conformational free energy distinguish loop conformations in proteins?

Limitations in protein homology modeling often arise from the inability to adequately model loops. In this paper we focus on the selection of loop conformations. We present a complete computational treatment that allows the screening of loop conformations to identify those that best fit a molecular model. The stability of a loop in a protein is evaluated via computations of conformational free energies in solution, i.e., the free energy difference between the reference structure and the modeled one. A thermodynamic cycle is used for calculation of the conformational free energy, in which the total free energy of the reference state (i.e., gas phase) is the CHARMm potential energy. The electrostatic contribution of the solvation free energy is obtained from solving the finite-difference Poisson-Boltzmann equation. The nonpolar contribution is based on a surface area-based expression. We applied this computational scheme to a simple but well-characterized system, the antibody hypervariable loop (complementarity-determining region, CDR). Instead of creating loop conformations, we generated a database of loops extracted from high-resolution crystal structures of proteins, which display geometrical similarities with antibody CDRs. We inserted loops from our database into a framework of an antibody; then we calculated the conformational free energies of each loop. Results show that we successfully identified loops with a "reference-like" CDR geometry, with the lowest conformational free energy in gas phase only. Surprisingly, the solvation energy term plays a confusing role, sometimes discriminating "reference-like" CDR geometry and many times allowing "non-reference-like" conformations to have the lowest conformational free energies (for short loops). Most "reference-like" loop conformations are separated from others by a gap in the gas phase conformational free energy scale. Naturally, loops from antibody molecules are found to be the best models for long CDRs (> or = 6 residues), mainly because of a better packing of backbone atoms into the framework of the antibody model.     

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.     

Complete relaxation and conformational exchange matrix (CORCEMA) analysis of NOESY spectra of interacting systems; two-dimensional transferred NOESY

A very general procedure entitled complete relaxation and conformational exchange matrix (CORCEMA) analysis has been developed to analyze the 2D-NOESY spectra of interacting systems undergoing multistate conformational exchange. This is an extension of earlier work from this laboratory on the methodological treatment of multistate conformational exchange [Krishna et al., Biopolymers 19, 2003 (1980)] and the theory of transferred NOESY for finite exchange off-rates [Lee and Krishna, J. Magn. Reson. 98, 36 (1992)]. The current theory is based on generalized rate matrices for relaxation and conformational exchange. The CORCEMA algorithm explicitly incorporates intermolecular dipolar cross relaxation between the molecules when they are complexed. It permits an analysis of NOESY intensities for the intra- as well as intermolecular contacts between the interacting molecules under a variety of binding conditions. Its application is illustrated on two examples of transferred NOESY simulations: (1) a two-state system involving a ligand and an enzyme forming a ligand-enzyme complex, and (2) a three-state system in which the ligand-enzyme complex can undergo a conformational transition from an "open state" to a "closed state," and can include conformational changes in both the complexed ligand and the complexed enzyme, such as hinge-bending motions. Simplifying expressions for generalized matrix analyses are derived for three limiting cases of the three-state system. This three-state example is illustrated using a hypothetical model of the hinge-bending motion in a thermolysin-inhibitor complex. It is shown that: (1) The neglect of cross relaxation between the interacting species in their complexed forms can lead to misleading conclusions on the "bound" conformation of the ligand. (2) If protein-mediated spin diffusion is dominant, caution is needed in analyses based on initial slopes alone due to one's inability to identify the exact range of the initial growth curve under poor signal/noise situations. (3) The neglect of conformational changes upon complexation, e.g., hinge-bending motions of the ligand-enzyme complex, can lead to erroneous results on the nature of "bound" conformations of the ligand. In this case, attempts to analyze the transferred NOESY data with a two-state model will result in a "virtual" conformation for the bound ligand. (4) When the hinge-bending rate is slower than the cross relaxation and enzyme off-rates, the bound conformation of a ligand deduced from the transferred NOESY experiment is more likely to represent nonspecific or weak binding in an open state of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of gamma-aminobutyric acid (GABA) transporters by extracellular GABA

Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.     

A stringent test for the nucleotide switch mechanism of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. The X-ray crystal structure of the enzyme has revealed that the two nucleotide binding sites are separated by approximately 35 A. Isotopic oxygen exchange of 18O and 16O between solvent water and [13C]bicarbonate was measured using 13C NMR spectroscopy during substrate turnover in the presence and absence of glutamine as a nitrogen source. In the absence of added glutamine, CPS catalyzed the exchange of one oxygen atom from bicarbonate with solvent water during every turnover of the bicarbonate-dependent ATPase reaction. In the presence of added glutamine, there was no exchange of solvent water with bicarbonate during the enzymatic synthesis of carbamoyl phosphate, indicating that any carbon-containing intermediate in the reaction mechanism is committed to the formation of carbamoyl phosphate and is not subject to hydrolysis. These results are fully consistent with a chemical mechanism that requires the physical migration of the carbamate intermediate from the site of its formation within one of the nucleotide binding domains to the other nucleotide binding domain for subsequent phosphorylation by the second MgATP. These results are not compatible with a nucleotide switch mechanism. The nucleotide switch mechanism includes the synthesis of carbamoyl phosphate entirely within a single nucleotide binding domain and concurrent conformational changes driven by the bicarbonate-dependent hydrolysis of MgATP at the second nucleotide binding domain.     

Ultraviolet difference spectroscopy study of alpha- and beta gamma-thrombin binding to heparin

The interaction of alpha- and beta gamma-thrombin with heparin was studied by ultraviolet difference spectroscopy within the wavelength range of 230-300 nm. The absorption difference spectrum of the thrombin-heparin complex was negative and had two maxima at 255 nm (5300 M-1 cm-1) and 282 nm (4700 M-1 cm-1) for alpha-thrombin and at 240 nm (4900 M-1 cm-1) and 282 nm (4100 M-1 cm-1) for beta gamma-thrombin. It is assumed that the conformational changes induced by heparin in the enzyme molecule involve the transfer of some tryptophan and tyrosine residues from the interior of the protein to the surface. The absorption changes during alpha-thrombin--heparin interaction at physiological ionic strength suggest binding of some alpha-thrombin molecules to a heparin molecule at the ligand-enzyme molar ratio lower than 1. Under the same conditions beta gamma-thrombin forms an equimolar complex with heparin with the dissociation constant equal to 7,0.10(-9) M. The ionic strength increase up to 0,217 M NaCl results in some disturbances in beta gamma-thrombin-heparin interaction and prevents the binding of additional alpha-thrombin molecules to an equimolar complex of alpha-thrombin with heparin. Therefore the kinetics of the two enzyme forms interaction with heparin are similar, the alpha-thrombin affinity for heparin being a little higher. The data obtained suggest that alpha-thrombin binding to heparin is essential for biological inactivation of thrombin.     

Study of yeast DNA topoisomerase II and its truncation derivatives by transmission electron microscopy

The 1429-amino acid residue long yeast DNA topoisomerase II and three of its deletion derivatives, a C-terminal truncation containing residues 1-1202, a 92-kDa fragment spanning residues 410-1202, and an A'-fragment spanning residues 660-1202, were examined by transmission electron microscopy. Analysis of rotary-shadowed images of these molecules shows that the full-length enzyme assumes a tripartite structure, in which a large globular core comprising the carboxyl parts of the dimeric enzyme is connected to a pair of smaller spherical masses comprising the ATPase domains of the enzyme. The linkers bridging the large globular structure and each of the smaller spheres are not visible in most of the images but appear to be sufficiently stiff to keep the relative positions of the connected parts. The angle extended by the pair of spherical masses is variable and falls in a range of 50-100 degrees for the majority of the images. On binding of a nonhydrolyzable ATP analog to the enzyme, this angle is significantly reduced as the two spherical masses swing into contact. These observations, together with results from previous biochemical and x-ray crystallographic studies of the enzyme, provide a sketch of the molecular architecture and conformational states of a catalytically active type II DNA topoisomerase.     

Conformational modifications of smooth muscle light chain kinase

Our recent investigations have shown that smooth muscle myosin light chain kinase (MLCK) exists in solution as a mixture of oligomeric, dimeric and monomeric species; besides during preincubation (maintaining of the activated enzyme without substrate) with substoichiometric amounts of calmodulin (CaM) it undergoes definite changes leading to several fold lowering of its activity. Fluorescent data obtained in this work suggest that such kinase inhibition must not be connected with quantitative redistribution of different kinase species but rather it is the result of conformational modifications of this enzyme activated molecules leading to the reduction of their affinity to CaM. Such conformational rearrangements took place also at equimolar kinase to CaM ratio (or CaM excess) but in this case they were characterized by lower depth and insignificant MLCK activity fall. The nature of these conformational changes is discussed.     

Optical changes in unilamellar vesicles experiencing osmotic stress

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.     

Catalysis of the hydrolysis of phosphorylated pyridines by alkaline phosphatase has little or no dependence on the pKa of the leaving group

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.     

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.     

Conformational analysis of [Met5]-enkephalin: solvation and ionization considerations

[Met5]-Enkephalin has the sequence Tyr-Gly-Gly-Phe-Met. Only the extended conformation of the peptide has been observed by X-ray crystallography. Nuclear magnetic resonance spectroscopy supports the presence of a turn at Gly 3 and Phe 4 in dimethyl sulfoxide. In this study, the peptide conformational states and thermodynamic properties are understood in terms of ionization state and solvent environment. In the calculation, final conformations obtained from multiple independent Monte Carlo simulated annealing conformational searches are starting points for molecular dynamics simulations. In an aqueous environment given by the use of solvation free energy and the zwitterionic state, dominant structural motifs computed are G-P Type II' bend, G-G Type II' bend, and G-G Type I' bend motifs, in order of increasing free energy. In the calculation of the peptide with neutral N- and C-termini and solvation free energy, the extended conformer dominates (by at least a factor of 2.5), and the conformation of another low free energy conformer superimposes well on the pharmacophoric groups of morphine. Neutralization of charge and solvation induce and stabilize the extended conformation, respectively. A mechanism of inter-conversion between the extended conformer and three bent conformers is supported by phi/psi-scatter plots, and by the conformer relative free energies. An estimate of the entropy change of receptor unbinding is 8.3 cal K-1 mol-1, which gives rise to a -2.5 kcal/mol entropy contribution to the free energy of unbinding at 25 degrees C. The conformational analysis methodology described here should be useful in studies on short peptides and flexible protein surface loops that have important biological implications.     

Interaction of glucose oxidase with phospholipid vesicles

The interactions between glucose oxidase and phospholipid vesicles were investigated. The investigations were carried on molecules adsorbed on the outer surfaces as well as entrapped in the interior of the vesicles . The adsorption of glucose oxidase on the surfaces of egg egg licithin vesicles, containing varying amounts of cholesterol and stearoylamine was measured by determining the free fraction of glucose oxidase detected in the filtrates. In general an enhancement of enzymic activity was observed upon interaction with the vesicles. The enhancement depends on the lipid composition of the vesicles and the surface concentration of the adsorbed glucose oxidase. It reached a maximal value at a surface concentration of 1.4-10(11) molecules/cm2 (approximately 7.1 - 10(4) A2/molecule) on pure phosphatidylcholine vesicles and about 6.5 - 10(10) molecules/cm2 (approximately 16 - 10(4) A2/molecule) when the vesicles contained cholesterol or cholesterol and stearoylamine. CD measurements indicated that the change in enzymic activity of the adsorbed glucose oxidase was accompanied by conformational modification of the enzyme. In order to entrap glucose oxidase into the vesicles, the lipid was sonicated in the presence of the enzyme. After removal of the free and adsorbed enzyme the amount of the entrapped enzyme was determined by measuring its activity after disintegration of the vesicles with Triton. The enzymic activity of the entrapped glucose oxidase served as a measure for the permeability of the bilayer membrane of the lipid vesicles to glucose. Addition of insulin to the suspension of vesicles containing the entrapped glucose oxidase increased the permeability of glucose by up to 9 - 10(-8) cm/s. This value is the lowest estimate based on the assumption that one glucose oxidase molecule was entrapped in every vesicle.     

Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril-ACE complex exhibits a heat protonation of approximately -30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn2+. Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding deltaG degrees is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, deltaCp =-4.3+/-0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative deltaCp indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand-protein interface, and slight conformational changes in the vicinity of the active sites.     

Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.     

Autoimmunity to collagen in human lung cancer

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.     

Visualisation of extensive water ribbons and networks in a DNA minor-groove drug complex

The crystal structure is reported of a complex between an ethyl derivative of the minor-groove drug furamidine and the dodecanucleotide duplex d(CGCGAATTCGCG)2, which has been refined to 1.85 A resolution and an R factor of 16.6% for data collected at -173 degreesC. An exceptionally large number (220) of water molecules have been located. The majority of these occur in the first coordination shell of solvation. There are extensive networks of connected waters, both in the major and minor grooves. In particular, there are 21 water molecules associated with the minor-groove drug, via hydrogen bonds from the four charged nitrogen atoms. One cluster of four waters is situated in the groove itself; the majority are on the outer edge of the groove, and serve to bridge between the outward-directed drug nitrogen atoms and backbone phosphate oxygen atoms. These bridges are both intra- and inter-strand, with the net effect that the outer edge of the drug molecule is covered by ribbons of water molecules.