Clustering of atomic displacement parameters in bovine trypsin reveals a distributed lattice of atoms with shared chemical properties.

Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments.

NMR structure of CmPI-II, a non-classical Kazal protease inhibitor: Understanding its conformational dynamics and subtilisin A inhibition.

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2' positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2') contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality.

The major Bowman-Birk inhibitor (BBIs) of horsegram (Dolichos biflorus) HGI-III, contains seven interweaving disulfides and is extremely stable to high temperatures. The contributions of two disulfide bonds in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants of wild type protein. Thermal denaturation kinetics, differential scanning calorimetry and urea denaturation studies indicate that the absence of either of the two disulfides destabilizes the protein significantly. C20-C66 contributes substantially to both thermal stability and controls trypsin and chymotrypsin inhibitor activity. These two disulfides act in synergy as deletion of both disulfides leads to a complete loss of thermal stability. The data indicate that the two subdomains are not entirely independent of each other. Long range interactions, between the domains are facilitated by C20-C66. The deletion of the disulfide bonds also increased proteolytic susceptibility in a manner similar to the decreased thermal stability. From this study of rHGI a prototype of legume BBIs in can be concluded that among the array of seven evolutionarily conserved disulfide bonds, the disulfide C20-C66 that connects a residue in the trypsin domain with a residue at the border of the same domain plays a dominant role in maintaining functional and structural stability.

Trypsin-gold nanoparticle conjugates: binding, enzymatic activity, and stability.

Nanomaterials may interact with biomolecules in various ways and change their bioactivities. Here, we report on how gold nanoparticles (AuNPs) affect a most important protease, trypsin. After simple mixing of the trypsin and AuNPs solution the average diameter is 20 nm under enzyme friendly conditions (pH 8.0); the bond between trypsin and AuNPs was detected by UV-vis spectroscopy. The further protease assay of trypsin, before and after mixing with the AuNPs solution, pointed out an improved performance in terms of enzyme activity and stability.

Covalent attachment of trypsin on plasma polymerized allylamine.

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV-vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.

Trypsin entrapped in poly(diallyldimethylammonium chloride) silica sol-gel microreactor coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An enzyme-immobilized capillary microreactor for rapid protein digestion and proteomics analysis is reported. The inner surface of the fused-silica capillary was coated with poly(diallyldimethylammonium chloride) (PDDA)-entrapped silica sol-gel matrix, followed by assembly of trypsin onto the PDDA-modified surface via electrostatic adsorption. The immobilization parameters such as PDDA content in the sol-gel matrix, trypsin concentration and pH were investigated in detail. Protein samples including beta-casein, myoglobin and cytochrome c could be effectively digested and electrophoretically separated simultaneously in such a modified capillary. Just 2.26 ng (corresponding to 0.10-0.14 picomole) of sample was sufficient for on-line capillary electrophoresis peptide mapping. The efficiency of the digestion was further demonstrated by digestion of a human liver cytoplasm sample and 253 proteins were identified in one unique run.

Two-center-multipole expansion method: application to macromolecular systems.

We propose a theoretical method for the calculation of the interaction energy between macromolecular systems at large distances. The method provides a linear scaling of the computing time with the system size and is considered as an alternative to the well-known fast multipole method. Its efficiency, accuracy, and applicability to macromolecular systems is analyzed and discussed in detail.

Electrostatic potentials of proteins in water: a structured continuum approach.

Electrostatic interactions play a crucial role in many biomolecular processes, including molecular recognition and binding. Biomolecular electrostatics is modulated to a large extent by the water surrounding the molecules. Here, we present a novel approach to the computation of electrostatic potentials which allows the inclusion of water structure into the classical theory of continuum electrostatics. Based on our recent purely differential formulation of nonlocal electrostatics [Hildebrandt, et al. (2004) Phys. Rev. Lett., 93, 108104] we have developed a new algorithm for its efficient numerical solution. The key component of this algorithm is a boundary element solver, having the same computational complexity as established boundary element methods for local continuum electrostatics. This allows, for the first time, the computation of electrostatic potentials and interactions of large biomolecular systems immersed in water including effects of the solvent's structure in a continuum description. We illustrate the applicability of our approach with two examples, the enzymes trypsin and acetylcholinesterase. The approach is applicable to all problems requiring precise prediction of electrostatic interactions in water, such as protein-ligand and protein-protein docking, folding and chromatin regulation. Initial results indicate that this approach may shed new light on biomolecular electrostatics and on aspects of molecular recognition that classical local electrostatics cannot reveal.

Synthesis of cross-linked enzyme aggregates (CLEAs) in CO2-expanded micellar solutions.

A new method to prepare the cross-linked enzyme aggregates (CLEAs) was developed. Through cross-linking the enzyme (Trypsin) aggregates, which was precipitated from the CO2-expanded reverse micellar solutions, dendritic CLEAs were obtained. The sizes of the CLEAs prepared by this new method were nanometer order of magnitudes and could be tuned by changing the water-to-surfactant ratio (w0) and the concentration of enzyme in the reverse micellar solution. The diameter of CLEAs increased with increasing w0 value of reverse micelles and the concentration of Trypsin. The activity of CLEAs obtained by this method is improved in contrast to those obtained by the conventional method. This method has some advantages in applications and can be easily applied to the synthesis of other cross-linked enzyme aggregates.

Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant.

Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.

Manufacturing of nanochannels with controlled dimensions using protease nanolithography.

The feasibility of creating nanometer scale depressions in biological substrates using active enzymes delivered with scanning probe microscopes has been previously demonstrated by us and other groups. Here we present a comprehensive study revealing the dependence of channels dimensions on the parameters of the "writing" process and provide a simple way to precisely control their dimensions. Such nanochannels may be used in nanofluidic biochip applications.

Crystal structure of Streptomyces erythraeus trypsin at 1.9 A resolution.

A trypsin-like serine protease from Streptomyces erythraeus (abbreviated as SET) has been crystallized at pH 7, which is within its active pH range. The crystal structure of SET has been solved by molecular replacement using the atomic model of Streptomyces griseus trypsin (SGT), which is 37% homologous with SET, and refined to the crystallographic R factor of 0.199 for 15,878 reflections with Fo/sigma(F) > 3 between 7 and 1.9 A resolution. The final model of SET contains 1,619 protein atoms and 97 water molecules. No Ca2+ ion is present in SET apparently because (i) the two carboxylate groups from two Glu residues, which bind a Ca2+ ion in bovine trypsin (BT) or SGT, have disappeared; and (ii) a guanidino group from an Arg residue is unfavorably present in the potential binding region. There is an unusual type II beta-turn in which the third residue is Asp instead of Gly. This Asp residue is the only non-Gly residue significantly outside the allowed regions in the Ramachandran map. The three-dimensional structure of SET is essentially the same as those of other trypsins of mammalian origin. The 211 C alpha atoms of SET exhibit an r.m.s. deviation of 1.16 A with equivalent atoms of BT, and the 208 equivalent C alpha atoms between SET and SGT exhibit an r.m.s. deviation of 1.09 A. The large deviations in C alpha positions between SET and BT or between SET and SGT are mainly observed in the first domain. The conformations of the side-chains of the catalytic triad are mutually similar to each other in these three proteases. Each of the chi 1 torsion angles of the three residues is distributed within +/- 5 degrees from each corresponding mean value. The hydrogen bond distances related to the side-chains in the triad coincide fairly well, though the relative disposition of the side-chains differs by 0.1-0.6 A among SET, BT, and SGT. The hydrogen bond network concerned with Ser(195), Asp(189), and water molecules in the substrate binding pocket differs from that in BT or SGT.

Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.

Geometry of binding of the benzamidine- and arginine-based inhibitors N alpha-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-pipe ridine (NAPAP) and (2R,4R)-4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid (MQPA) to human alpha-thrombin. X-ray crystallographic determination of the NAPAP-trypsin complex and modeling of NAPAP-thrombin and MQPA-thrombin.

The X-ray crystal structure of the trypsin complex formed with N alpha-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piper idine (NAPAP) was determined with X-ray data to 0.18-nm resolution and crystallographically refined. NAPAP binds into the active site of trypsin in a quite compact form: the p-amidinophenylalanine moiety of the D-stereoisomer binds into the specificity pocket; the glycyl group is hydrogen bonded with Gly216; the naphthyl group stands perpendicular to the indole moiety of Trp215; the piperidine ring is tightly packed between this naphthyl moiety and His57; in consequence the carboxy-terminal amido bond of NAPAP is located in such a way that it is not susceptible to the active-site Ser195. NAPAP and (2R,4R)-4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid (MQPA) [Matzusaki, T., Sasaki, C., Okumura, C. & Umeyama (1989) J. Biochem. (Tokyo) 105, 949-952] were transferred in their trypsin-binding conformations to human alpha-thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R. & Hofsteenge, J. (1989) EMBO J. 8. 3467 - 3475] and energy minimized. Both synthetic inhibitors fit perfectly into the much more restricted active site of thrombin. The accommodation of the S-aryl moieties in the 'aryl-binding site' and of the piperidine rings in the S2 subsite of thrombin are particularly favorable. The preference of thrombin for distinctly substituted piperidine derivatives and its generally higher (compared with trypsin) affinity for benzamidine and arginine-based inhibitors can be accounted for by these thrombin inhibitor models.

A novel approach to prediction of the 3-dimensional structures of protein backbones by neural networks.

Three-dimensional structures of protein backbones have been predicted using neural networks. A feed forward neural network was trained on a class of functionally, but not structurally, homologous proteins, using backpropagation learning. The network generated tertiary structure information in the form of binary distance constraints for the C(alpha) atoms in the protein backbone. The binary distance between two C(alpha) atoms was 0 if the distance between them was less than a certain threshold distance, and 1 otherwise. The distance constraints predicted by the trained neural network were utilized to generate a folded conformation of the protein backbone, using a steepest descent minimization approach.