Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor.

The structure of BPTI and reduced BPTI in concentrated guanidinium HCl (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Interprobe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its epsilon-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 +/- 2 A) in the native state. Upon unfolding by reduction with DTT or beta-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced average interprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted beta sheet element (residues 18-35), increased upon unfolding. At -30 degrees C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 +/- 20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 +/- 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even under denaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded proteins and for search for folding initiation sites (FISs) and early folding intermediates.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

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.     

Distributions of intramolecular distances in the reduced and denatured states of bovine pancreatic ribonuclease A. Folding initiation structures in the C-terminal portions of the reduced protein

The purpose of this investigation is to characterize the reduced state of RNase A (r-RNase A) in terms of (i) intramolecular distances, (ii) the sequence of formation of stable loops in the initial stages of folding, and (iii) the unfolding transitions induced by GdnHCl. This is accomplished by identifying specific subdomain structures and local and long-range interactions that direct the folding process of this protein and lead to the native fold and formation of the disulfide bonds. Eleven pairs of dispersed sites in the RNase A molecule were labeled with fluorescent donor and acceptor probes, and the distributions of intramolecular distances (IDDs) were determined by means of time-resolved dynamic nonradiative excitation energy transfer (TR-FRET) measurements. The mutants were designed to search for (a) a possible nonrandom fold of the backbone in the collapsed state and (b) possible loops stabilized by long-range interactions. It was found that, under folding conditions, (i) the labeled mutants of r-RNase A in refolding buffer (the R(N) state) exhibit features of specific (nonrandom) compact but very dispersed subdomain structures (indicated by short mean distances, broad IDDs, and a weak dependence of the mean distances on segment length), (ii) the backbone fold in the C-terminal beta-like portion of the molecule appears to adopt a native-like overall fold, (iii) the N-terminal alpha-like portion of the chain is separated from the C-terminal core by very large intramolecular distances, larger than those in the crystal structure, and (iv) perturbations by addition of GdnHCl reveal several conformational transitions in different sections of the chain. Addition of GdnHCl to the native disulfide-intact protein provided a reference state for the extent of expansion of intramolecular distances under denaturing conditions. In conclusion, r-RNase A under folding conditions (the R(N) state) is poised for the final folding step(s) with a native-like trace of the chain fold but a large separation between the two subdomains which is then decreased upon introduction of three of the four native disulfide cross-links.     

Statistical measures on residue-level protein structural properties

The atomic-level structural properties of proteins, such as bond lengths, bond angles, and torsion angles, have been well studied and understood based on either chemistry knowledge or statistical analysis. Similar properties on the residue-level, such as the distances between two residues and the angles formed by short sequences of residues, can be equally important for structural analysis and modeling, but these have not been examined and documented on a similar scale. While these properties are difficult to measure experimentally, they can be statistically estimated in meaningful ways based on their distributions in known proteins structures. Residue-level structural properties including various types of residue distances and angles are estimated statistically. A software package is built to provide direct access to the statistical data for the properties including some important correlations not previously investigated. The distributions of residue distances and angles may vary with varying sequences, but in most cases, are concentrated in some high probability ranges, corresponding to their frequent occurrences in either α-helices or β-sheets. Strong correlations among neighboring residue angles, similar to those between neighboring torsion angles at the atomic-level, are revealed based on their statistical measures. Residue-level statistical potentials can be defined using the statistical distributions and correlations of the residue distances and angles. Ramachandran-like plots for strongly correlated residue angles are plotted and analyzed. Their applications to structural evaluation and refinement are demonstrated. With the increase in both number and quality of known protein structures, many structural properties can be derived from sets of protein structures by statistical analysis and data mining, and these can even be used as a supplement to the experimental data for structure determinations. Indeed, the statistical measures on various types of residue distances and angles provide more systematic and quantitative assessments on these properties, which can otherwise be estimated only individually and qualitatively. Their distributions and correlations in known protein structures show their importance for providing insights into how proteins may fold naturally to various residue-level structures.     

Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations.

Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.     

Importance of long-range interactions in protein folding

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts. The residues occurring in the range of 4-10 residues apart contribute more towards long-range contacts in all-alpha proteins while the range is 11-20 in all-beta proteins. The hydrophobic residues Cys, Ile and Val prefer the 11-20 range and all other residues prefer the 4-10 range. The residues in all-beta proteins have an average of 3-8 long-range contacts whereas the residues in other classes have 1-4 long-range contracts. Furthermore, the preference of residue pairs to the folding and stability will be discussed.     

Formation of the hydrophobic core of ribonuclease A through sequential coordinated conformational transitions

With steady-state and time-resolved fluorescence energy-transfer measurements, we determined the distributions of intramolecular distances in nine mutants to study the conformations of wild-type ribonuclease A in the reduced state under folding conditions. Although far-UV-CD measurements show no evidence for a secondary-structure transition, temperature- and GdnHCl-induced changes in intramolecular distance distributions in the reduced state revealed evidence for long-range subdomain structures in the denatured protein. These poorly defined structures, reflected here by wide distributions corresponding to a wide range of energies, form during refolding in a complex sequence of multiple subdomain transitions. A more well-defined structure emerges only when this structural framework, which directs the successive steps in the folding process, matures and is reinforced by stronger interactions such as disulfide bonds.     

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers (n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as "BPTI decamer" crystals.     

Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.

Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.     

The concentration dependence of the diffusion coefficient for bovine pancreatic trypsin inhibitor: a dynamic light scattering study of a small protein

This paper reports the use of dynamic light scattering to investigate the concentration dependence of the diffusion coefficient for bovine pancreatic trypsin inhibitor (BPTI). BPTI is a small molecular weight protein (6511 Da) that has been the subject of numerous experimental studies. In addition to addressing questions that remain in the literature concerning the aggregation behavior of BPTI, we show that dynamic light scattering can be practically applied to proteins as small as BPTI, and that it can provide a useful means of parameterizing the solution behavior for proteins. We obtained values for the apparent diffusion coefficient of BPTI as a function of concentration over a range of pH values from 2.59 to 9.92 at an ionic strength of 0.3M, and over a range of ionic strength values from 0.1 to 0.5M at a pH of 7.0. The concentration dependence is linear for nearly all the conditions examined, even up to concentrations as high as 65 mg/mL. The average diffusion coefficient obtained at infinite dilution is 14.4 +/- 0.2 x 10(-7) cm2/s. This value agrees with that expected for a BPTI monomer hydrated with less than a monolayer of water. We used the theories of Felderhof, of Batchelor, and of Phillies, along with the DLVO theory to interpret the concentration dependence of the apparent diffusion coefficient. The variations observed with pH and ionic strength can be primarily attributed to screened coulombic interactions. In addition, there is an attractive interaction that is slightly stronger than the repulsive coulombic one, and that is essentially independent of pH and ionic strength. The attractive interactions appear to arise from nonspecific van der Waals interactions and do not lead to the formation of stable aggregates of BPTI.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Residual structure in unfolded proteins

The denatured state ensemble (DSE) of unfolded proteins, once considered to be well-modeled by an energetically featureless random coil, is now well-known to contain flickering elements of residual structure. The position and nature of DSE residual structure may provide clues toward deciphering the protein folding code. This review focuses on recent advances in our understanding of the nature of DSE collapse under folding conditions, the quantification of the stability of residual structure in the DSE, the determination of the location and types of residues involved in thermodynamically significant residual structure and advances in detection of long-range interactions in the DSE.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.     

Insight into the factors influencing the backbone dynamics of three homologous proteins,dendrotoxins I and K,and BPTI: FTIR and time-resolved fluorescence investigations

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties.     

Effects of limited input distance constraints upon the distance geometry algorithm.

In this paper we examine the distance geometry (DG) algorithm in the form used to determine the structure of proteins. We focus on three aspects of the algorithm: bound smoothing with the triangle inequality, the random selection of distances within the bounds, and the number of distances needed to specify a structure. Computational experiments are performed using simulated and real data for basic pancreatic trypsin inhibitor (BPTI) from nmr and crystallographic measurements. We find that the upper bounds determined by bound smoothing to be a linear function of the true crystal distance. A simple model that describes the results obtained with randomly selected trial distances is proposed. Using this representation of the trial distances, we show that BPTI DG structures are more compact than the true crystal structure. We also show that the DG-generated structures no longer resemble test structures when the number of these interresidue distance constraints is less than the number of degrees of freedom of the protein backbone. While the actual model will be sensitive the way distances are chosen, our conclusions are likely to apply to other versions of the DG algorithm.     

Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: application of long-range order to folding rate prediction.

The contact order is believed to be an important factor for understanding protein folding mechanisms. In our earlier work, we have shown that the long-range interactions play a vital role in protein folding. In this work, we analyzed the contribution of long-range contacts to determine the folding rate of two-state proteins. We found that the residues that are close in space and are separated by at least ten to 15 residues in sequence are important determinants of folding rates, suggesting the presence of a folding nucleus at an interval of approximately 25 residues. A novel parameter "long-range order" has been proposed to predict protein folding rates. This parameter shows as good a relationship with the folding rate of two-state proteins as contact order. Further, we examined the minimum limit of residue separation to determine the long-range contacts for different structural classes. We observed an excellent correlation between long-range order and folding rate for all classes of globular proteins. We suggest that in mixed-class proteins, a larger number of residues can serve as folding nuclei compared to all-alpha and all-beta proteins. A simple statistical method has been developed to predict the folding rates of two-state proteins using the long-range order that produces an agreement with experimental results that is better or comparable to other methods in the literature.     

The relation of polypeptide hormone structure and flexibility to receptor binding: The relevance of X-ray studies on insulins,glucagon and human placental lactogen

Summary This study is concerned with nondenaturational structural rearrangements of proteins in solution under the influence of physiologically moderate temperatures and salts.Temperature-induced rearrangements are viewed as the reason for breaks in Arrhenius curves of the enzymatic activity. In the cytosol as well as in biological membranes, proteins remain conformationally labile and participate in cooperative structural transitions of membranes. Such transitions are initiated by physiologically moderate temperatures, hormones, salts and aminoacids and affect the functional activity of cell membranes. It is suggested that structural lability of proteins and membranes is of importance in metabolic regulation.It may be said without any exaggeration that a basic objective of biochemistry and biophysics is to find the mechanisms by which coordination of numerous chemical and physicochemical processes along with adaptation to a changing environment can be regulated by the cell. An analysis of a large body of accumulated material and information on this subject leads us to a simple idea; namely, that metabolism is regulated primarily through weak physicochemical interactions. It is weak bonds arising at the sites of contact between effector and regulated macromolecules which serve as a trigger for the regulatory mechanisms of various types. This principle is fundamental for the long range mechanism and for the short-range cytoplasmic allosteric enzyme regulation. In each regulatory act of this type the regulated macromolecule undergoes conformational transition between the states of different functional activity.It is generally recognized^1,2 that in most cases conformational transition is cooperative by nature. However, biopolymers in a cell are an integral part of compact and orderly membraneous phases with active intermolecular interactions. Therefore it is pertinent to inquire whether the elementary act of regulation is necessarily always restricted to one macromolecule or whether there is a possibility of functionally important cooperative transition involving most if not all components of a polymolecular ensembles. We have in mind here the structural long-range effects when local perturbations in the receptor region of the membrane are able to propagate their effects to comparatively large distances. This would occur in a stepwise cooperative transition between two discrete structural states. After we expressed this idea^3,4 we discovered that there had been earlier opinions along such lines⁵ and in recent years similar views have become widely known^6,7,8.Since 1965 our laboratory has been concerned with experimental development of a hypothesis of the membraneous-cooperative-conformational mechanism in the regulation of life processes. We have taken the following path: nondenaturational conformational transitions of proteins in solution rearrangements at the isolated membrane level rearrangements in the intact membrane system of the cells.an invited article.