NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Limited sampling of conformational space by the distance geometry algorithm: implications for structures generated from NMR data

Calculations with a metric matrix distance geometry algorithm were performed that show that the standard implementation of the algorithm generally samples a very limited region of conformational space. This problem is most severe when only a small amount of distance information is used as input for the algorithm. Control calculations were performed on linear peptides, disulfide-linked peptides, and a double-stranded DNA decamer where only distances defining the covalent structures of the molecules (as well as the hydrogen bonds for the base pairs in the DNA) were included as input. Since the distance geometry algorithm is commonly used to generate structures of biopolymers from distance data obtained from NMR experiments, simulations were performed on the small globular protein basic pancreatic trypsin inhibitor (BPTI) that mimic calculations performed with actual NMR data. The results on BPTI and on the control peptides indicate that the standard implementation of the algorithm has two main problems: first, that it generates extended structures; second, that it has a tendency to consistently produce similar structures instead of sampling all structures consistent with the input distance information. These results also show that use of a simple root-mean-square deviation for evaluating the quality of the structures generated from NMR data may not be generally appropriate. The main sources of these problems are identified, and our results indicate that the problems are not a fundamental property of the distance geometry algorithm but arise from the implementations presently used to generate structures from NMR data. Several possible methods for alleviating these problems are discussed.     

Helix formation in model peptides based on nucleolin TPAKK motifs

The structures formed by peptide models of the N-terminal domain of the nucleolar protein nucleolin were studied by CD and nmr. The sequences of the peptides are based on the putative nucleic acid binding sequence motif TPAKK: The peptides TP1 and TP2 have the sequence acetyl-G(ATPAKKAA)_nG-amide, with n = 1 and 2, respectively. CD measurements indicate structural changes in both peptides when the lysine side chains are uncharged by increasing the pH or acetylation of the side-chain amines. When trifluoroethanol (TFE) is added, more extensive structural changes are observed, resembling helical structure based on nmr nuclear Overhauser effect (NOE) and C^α proton chemical shift changes, and CD spectra. The structure formed in 0.5M NaClO₄ as observed by nmr is similar to that when the lysine side chains are acetylated, due presumably to interactions of perchlorate ion with side-chain charges on lysines. The helical structure observed in TPAKK motifs may be stabilized via N-capping interactions involving threonine. The structures observed in TFE suggest that the Thr-Pro sequence initiates short helical segments in TPAKK motifs, and these helical structures might interact with nucleic acids, presumably via interactions between lysines and threonines of nucleolin. © 1995 John Wiley & Sons, Inc.     

Two-dimensional infrared population transfer spectroscopy for enhancing structural markers of proteins

We propose the possibility of using vibrational population transfer to enhance the structural markers for protein motifs that occur in two-dimensional infrared spectroscopy. We demonstrate the potential of this method by calculating the spectrum of the trpzip2 β-hairpin peptide, a system that is small enough to allow accurate simulation of its two-dimensional infrared spectra, including vibrational population transfer induced by a fluctuating solvent. The results show that under selected experimental conditions, in particular by using perpendicular polarization and finite waiting times, the cross peaks that constitute the well-known Z-shape marker for β-sheet structure in two-dimensional spectra are strongly enhanced. This enhancement is shown to result from vibrational population transfer. It should be possible to use the same technique for enhancing cross peaks in other structures and generally improve structure determination by two-dimensional infrared spectroscopy. The simulated population transfer times are in good agreement with those observed in experiments on typical proteins.     

Selective Bridging of Bis-Cysteinyl Residues by Arsonous Acid Derivatives as an Approach to the Characterization of Protein Tertiary Structures and Folding Pathways by Mass Spectrometry

Bis-cysteine selective modifications were successfully applied with melarsen oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and proteins were amenable to direct characterizations by mass spectrometry, e.g., direct molecular weight determinations and mass spectrometric peptide mapping that identified stoichiometry and sites of modification, respectively. Proteolytic digestion and mass spectrometric fragmentation of modified oxytocin showed that MEL-bridged peptide derivatives are structural homologues to the disulfide-bonded macrocyclic peptides. Mass spectrometric analyses determined the MEL modification site in partially reduced and selectively modified bovine pancreatic trypsin inhibitor (BPTI) bridging Cys-14 and Cys-38. The BPTI · MEL derivative was resistant to proteolysis by both Lys-C and trypsin and thus represented a rigid structure like native BPTI. MEL exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups, providing detailed tertiary structure information; (ii) high solubility as monomeric ortho acid in aqueous and organic solutions; (iii) adding a relatively large mass increment to proteins upon single modification; (iv) enabling UV monitoring of the derivatization due to a strong chromophor; and (v) performing fast and specific modifications of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of MEL. The investigated physical and chemical properties of MEL suggest general applicability for selective bis-thiol modifications, enabling protein structure–function studies in both soluble and membrane proteins and the study of protein-folding reactions.     

Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN

A set of conformational restraints derived from nuclear magnetic resonance (n.m.r.) measurements on solutions of the basic pancreatic trypsin inhibitor (BPTI) was used as input for distance geometry calculations with the programs DISGEO and DISMAN. Five structures obtained with each of these algorithms were systematically compared among themselves and with the crystal structure of BPTI. It is clear that the protein architecture observed in single crystals of BPTI is largely preserved in aqueous solution, with local structural differences mainly confined to the protein surface. The results confirm that protein conformations determined in solution by combined use of n.m.r. and distance geometry are a consequence of the experimental data and do not depend significantly on the algorithm used for the structure determination. The data obtained further provide an illustration that long intramolecular distances in proteins, which are comparable with the radius of gyration, are defined with high precision by relatively imprecise nuclear Overhauser enhancement measurements of a large number of much shorter distances.     

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg²⁺ levels. Populations treated with BPTI had fewer cells in S‐phase of the cell cycle and a corresponding increase of cells in G₀/G₁ and G₂ phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.     

Variable-Target-Function and Build-up Procedures for the Calculation of Protein Conformation. Application to Bovine Pancreatic Trypsin Inhibitor Using Limited Simulated Nuclear Magnetic Resonance Data

Abstract

An implementation of the variable-target-function procedure, first introduced by Braun and Gō [W. Braun and N. Gō, J. Mol. Biol. 186, 611–626 (1985)], has been used to generate conformations of the small protein bovine pancreatic trypsin inhibitor (BPTI), given a limited set of simulated data that could be obtained by nuclear magnetic resonance (NMR) techniques. A hybrid strategy was also used to calculate conformations of BPTI, given the same information. In the hybrid strategy, low-energy structures of medium-size fragments (decapeptides) of BPTI were generated using the variable-target-function method, followed by restrained energy optimization. The low-energy conformations were used as a basis to build up the complete fifty-eight-residue BPTI molecule. By using the variable-target-function approach, in which energy considerations were not introduced until full conformations of the entire BPTI molecule had been generated, it was not possible to obtain calculated structures with rms deviations from the X-ray conformation of less than 1.6 Å for the α-carbons. On the other hand, with the hybrid strategy, which involved the consideration of realistic energy terms in the early stages of the calculations, it was possible to calculate low-energy conformations of BPTI with rms deviations from the X-ray structure of 1.06 to 1.50 Å for the α-carbons. When the rms deviations were computed along the amino acid sequence, it was found that there was a good correlation between deviations among the calculated structures and deviations from the X-ray structure.     

Automated assignment of NOESY NMR spectra using a knowledge based method (KNOWNOE)

Automated assignment of NOESY spectra is a prerequisite for automated structure determination of biological macromolecules. With the program KNOWNOE we present a novel, knowledge based approach to this problem. KNOWNOE is devised to work directly with the experimental spectra without interference of an expert. Besides making use of routines already implemented in AUREMOL, it contains as a central part a knowledge driven Bayesian algorithm for solving ambiguities in the NOE assignments. These ambiguities mainly arise from chemical shift degeneration which allows multiple assignments of cross peaks. Using a set of 326 protein NMR structures, statistical tables in the form of atom-pairwise volume probability distributions (VPDs) were derived. VPDs for all assignment possibilities relevant to the assignments of interproton NOEs were calculated. With these data for a given cross peak with N possible assignments A _i(i = 1,...,N) the conditional probabilities P(A _i, a|V ₀) can be calculated that the assignment A _idetermines essentially all (a-times) of the cross peak volume V ₀. An assignment A _kwith a probability P(A _k, a|V ₀) higher than 0.8 is transiently considered as unambiguously assigned. With a list of unambiguously assigned peaks a set of structures is calculated. These structures are used as input for a next cycle of iteration where a distance threshold D _maxis dynamically reduced. The program KNOWNOE was tested on NOESY spectra of a medium size protein, the cold shock protein (TmCsp) from Thermotoga maritima. The results show that a high quality structure of this protein can be obtained by automated assignment of NOESY spectra which is at least as good as the structure obtained from manual data evaluation.     

Solution structure of a D,L-alternating oligonorleucine as a model of double-stranded antiparallel beta-helix

Conformational characteristics of alternating D,L linear peptides are of particular interest because of their capacity to form transmembrane channels with different transport properties, as some natural antibiotics do. Single- and double-stranded beta-helical structures are common for alternating D,L peptides. The stability of the beta-helix depends on several structural factors, such as the backbone peptide length, type and position of side chains, and nature of terminal groups. The NMR and molecular dynamics solution conformation of a synthetic alternating D,L-oligopeptide with 15 norleucines (XVMe) has been used as a model to get insight in to the conformational features of double-stranded beta-helix structures. The NH chemical shift values (delta(NH)) and long-range nuclear Overhauser effects (NOE) cross peaks, in particular interstrand connectivities, clearly point to an antiparallel double-stranded beta-helix for the XVMe major conformation in solution. An extensive set of distances (from NOE cross peaks) and H-bonds (from delta(NH)) has been included in the molecular dynamics calculations. The experimental NMR data and theoretical calculations clearly indicate that the most probable conformation of XVMe in solution is a double-strand antiparallel beta(5.6) increasing decreasing-helix structure.     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Nuclear overhauser effect spectroscopy (NOESY) detection of the specific interaction between substituents in cellulose and amylose triacetates

Nuclear Overhauser effect spectroscopy measurements on cellulose triacetate and on amylose triacetate with a mixing time of 500 ms, on the order of T₁, of acetyl protons, were performed to detect the specific through-space interaction between acetyl groups arising from their helix structures in solution. For cellulose triacetate, cross peaks were detected in CDCl₃ between acetyl proton signals at 3 and 6 positions on an anhydroglucose unit. In DMSO-d⁶, on the other hand, correlation peaks were observed not only between the 3 and 6 positions but also the 2 and 6 positions. For amylose triacetate, cross peaks were detected in CDCl₃ between the acetyl proton signals at the 2 and 6 positions. The through-space interaction of acetyl groups in cellulose triacetate and in amylose triacetate in solution was then interpreted based on their three-dimensional structures in solid state determined by x-ray crystallography. © 1994 John Wiley & Sons, Inc.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.     

BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FX_a (blood coagulation factor X_a protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FX_a target sequence allows specific cleavage of the hybrid protein. Effective FX_a cleavage is achieved by spacing the FX_a target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FX_a cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FX_a specificity. The yield of highly purified recombinant BPTI is 3–6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. ¹H NMR is used to analyze the N-extended BPTI analogues.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

An Effective Solvation Term Based on Atomic Occupancies for Use in Protein Simulations

Abstract

Several approaches to the treatment of solvent effects based on continuum models are reviewed and a new method based on occupied atomic volumes (occupancies) is proposed and tested. The new method describes protein-water interactions in terms of atomic solvation parameters, which represent the solvation free energy per unit of volume. These parameters were determined for six different atoms types, using experimental free energies of solvation. The method was implemented in the GROMOS and PRESTO molecular simulation program suites. Simulations with the solvation term require 20-50% more CPU time than the corresponding vacuum simulations and are approximately 20 times faster than explicit water simulations. The method and parameters were tested by carrying out 200 ps simulations of BPTI in water, in vacuo, and with the solvation term. The performance of the solvation term was assessed by comparing the structures and energies from the solvation simulations with the equivalent quantities derived from several BPTI crystal structures and from the explicit water and vacuum simulations. The model structures were evaluated in terms of exposed total surface, buried and exposed polar surfaces, secondary structure preservation, number of hydrogen bonds, energy contributions, and positional deviations from BPTI crystal structures. Vacuum simulations produced unrealistic structures with respect to all criteria applied. The structures resulting from the simulations with explicit water were closer to the 5PTI crystal structure, although part of the secondary structure dissolved. The simulations with the effective solvation term produce structures that are normal according to all evaluations and in most respects are remarkably similar to the 5PTI crystal structure despite considerable positional fluctuations during the simulations. The segments where the model and crystal structures differ are known to be flexible and the observed difference may be physically realistic. The effective solvation term based on occupancies is not only very efficient in terms of computer time but also results in meaningful structural properties for BPTI. It may therefore be generally useful in molecular dynamics of macromolecules.     

Simple method to introduce an ester infrared probe into proteins

The ester carbonyl stretching vibration has recently been shown to be a sensitive and convenient infrared (IR) probe of protein electrostatics due to the linear dependence of its frequency on local electric field. While an ester moiety can be easily incorporated into peptides via solid‐phase synthesis, currently there is no method available to site‐specifically incorporate it into a large protein. Herein, we show that it is possible to use a cysteine alkylation reaction to achieve this goal and demonstrate the feasibility of this simple method by successfully incorporating a methyl ester group (? CH₂COOCH₃) into a model peptide (YGGCGG), two amyloid‐forming peptides derived from the insulin B chain and Aβ, and bovine serum albumin (BSA). IR results obtained with those peptide and protein systems further confirm the utility of this vibrational probe in monitoring, for example, the structural integrity of amyloid fibrils and ligand binding‐induced changes in protein local hydration status.     

Molecular dynamics of the P450cam–Pdx complex reveals complex stability and novel interface contacts

Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5‐exo‐hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe₂S₂ containing protein. Two recent crystal structures of the P450cam–Pdx complex, one solved with the aid of covalent cross‐linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross‐linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross‐linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251–Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251–Arg186 ion pair may be important.     

Molecular dynamics simulation of solvated protein at high pressure.

We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)