Immunoglobulin-binding Domain Of Streptococcal Protein Research Articles

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid β42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

Fast NMR method to probe solvent accessibility and disordered regions in proteins

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

Purification of chicken IgY by binding capture using elastin-like polypeptide-tagged immunoglobulin-binding domain of streptococcal protein G

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin, but its practical application is limited due to the complex purification procedure. In this study, the C2 domain of streptococcal protein G (SPG) with the binding affinity for chicken IgY was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein, and purified to a high purity by inverse transition cycling (ITC). Binding experiments showed that chicken IgY could bind to and eluted off the ELP-C2 fusion protein in pH-, temperature- and/or time-dependent manners. By using the ELP-C2 protein, a simple chicken IgY purification method was developed, and its purification performance was compared with that of ammonium sulfate precipitation and ethanol fractionation. Quantitative SDS-PAGE analysis showed that the ELP-C2 binding capture method provided a chicken IgY purity of 96.3% and a recovery of 64%, both of which were significantly higher than that of the two traditional methods The ELP-C2 binding capture method could be accomplished within 3h, in contrast to 30.3h for ammonium sulfate precipitation or 4.3h for ethanol fractionation. These data suggest that the ELP-C2 binding capture was a simple, efficient and cost-effective method for purification of chicken IgY.

Polar Versus Non-polar Local Ordering at Mobile Sites in Proteins: Slowly Relaxing Local Structure Analysis of (15)N Relaxation in the Third Immunoglobulin-Binding Domain of Streptococcal Protein G.

We developed recently the slowly relaxing local structure (SRLS) approach for studying restricted motions in proteins by NMR. The spatial restrictions have been described by potentials comprising the traditional L = 2, K = 0, 2 spherical harmonics. However, the latter are associated with non-polar ordering whereas protein-anchored probes experience polar ordering, described by odd-L spherical harmonics. Here we extend the SRLS potential to include the L = 1, K = 0, 1 spherical harmonics and analyze (15)N-(1)H relaxation from the third immunoglobulin-binding domain of streptococcal protein G (GB3) with the polar L = 1 potential (coefficients c0(1) and c1(1)) or the non-polar L = 2 potential (coefficients c0(2) and c2(2)). Strong potentials, with ⟨c0(1)⟩ ∼ 60 for L = 1 and ⟨c0(2)⟩ ∼ 20 for L = 2 (in units of kBT), are detected. In the α-helix of GB3 the coefficients of the rhombic terms are c1(1) ∼ c2(2) ∼ 0; in the preceding (following) chain segment they are ⟨c1(1)⟩ ∼ 6 for L = 1 and ⟨c2(2)⟩ ∼ 14 for L = 2 (⟨c1(1)⟩ ∼ 3 for L = 1 and ⟨c2(2)⟩ ∼ 7 for L = 2). The local diffusion rate, D2, lies in the 5 × 10(9)-1 × 10(11) s(-1) range; it is generally larger for L = 1. The main ordering axis deviates moderately from the N-H bond. Corresponding L = 1 and L = 2 potentials and probability density functions are illustrated for residues A26 of the α-helix, Y3 of the β1-strand, and L12 of the β1/β2 loop; they differ considerably. Polar/orientational ordering is shown to be associated with GB3 binding to its cognate Fab fragment. The polarity of the local ordering is clearly an important factor.

NMR spin relaxation in proteins: The patterns of motion that dissipate power to the bath

We developed in recent years the two-body coupled-rotator slowly relaxing local structure (SRLS) approach for the analysis of NMR relaxation in proteins. The two bodies/rotators are the protein (diffusion tensor D1) and the spin-bearing probe, e.g., the 15N−1H bond (diffusion tensor, D2), coupled by a local potential (u). A Smoluchowski equation is solved to yield the generic time correlation functions (TCFs), which are sums of weighted exponentials (eigenmodes). By Fourier transformation one obtains the generic spectral density functions (SDFs) which underlie the experimental relaxation parameters. The typical paradigm is to characterize structural dynamics in terms of the best-fit values of D1, D2, and u. Additional approaches we pursued employ the SRLS TCFs, SDFs, or eigenmodes as descriptors. In this study we develop yet another perspective. We consider the SDF as function of the angular velocity associated with the fluctuating fields underlying NMR relaxation. A parameter called j-fraction, which represents the relative contribution of eigenmode, i, to a given value of the SDF function at a specific frequency, ω, is defined. j-fraction profiles of the dominant eigenmodes are derived for 0 ≤ ω ≤ 1012 rad/s. They reveal which patterns of motion actuate power dissipation at given ω-values, what are their rates, and what is their relative contribution. Simulations are carried out to determine the effect of timescale separation, D1/D2, axial potential strength, and local diffusion axiality. For D1/D2 ≤ 0.01 and strong local potential of 15 kBT, power is dissipated by global diffusion, renormalized (by the strong potential) local diffusion, and probe diffusion on the surface of a cone (to be called cone diffusion). For D1/D2 = 0.1, power is dissipated by mixed eigenmodes largely of a global-diffusion-type or cone-diffusion-type, and a nearly bare renormalized-local-diffusion eigenmode. For D1/D2 > 0.1, most eigenmodes are of a mixed type. The analysis is affected substantially by reducing the potential strength from 15 to 5 kBT, and/or allowing for axial D2 with D2,∥/D2,⊥ = 10. The scheme developed is applied to 15N−1H relaxation from the β-sheet residue K19 and the α-helix residue A34 of the third immunoglobulin-binding domain of streptococcal protein G. Previous studies revealed rhombic local potentials with different rhombicity around ${\rm C}_{i - 1}^\alpha {\rm - C}_i^\alpha $Ci−1α−Ciα, and different timescale separation (0.047 for K19 and 0.102 for A34). Here, we find that K19 and A34 dissipate power to the bath through global diffusion, mixed cone-diffusion-related and mixed renormalized-local-diffusion-related motions. At small ω-values, A34 is more effective than K19 in dissipating power. In general, it executes faster cone-diffusion-type, and slower renormalized-local-diffusion-type and local-probe-fluctuation-type motions. K19 experiences faster N−H fluctuations than A34. Eigenmode clustering, experienced by K19 to a larger extent, is observed in the fast-probe-fluctuation regime. New information on the effect of the structural context on N−H bond dynamics has been obtained. The patterns of motion that dissipate NMR-relaxation-related power illuminate protein dynamics from a new perspective. They constitute yet another qualifier of N−H bond dynamics. This study sets the stage for developing ways for enhancing the contribution of desired pathways for power dissipation at selected angular velocities.

The eigenmode perspective of NMR spin relaxation in proteins

We developed in recent years the two-body (protein and probe) coupled-rotator slowly relaxing local structure (SRLS) approach for elucidating protein dynamics from NMR spin relaxation. So far we used as descriptors the set of physical parameters that enter the SRLS model. They include the global (protein-related) diffusion tensor, D1, the local (probe-related) diffusion tensor, D2, and the local coupling∕ordering potential, u. As common in analyzes based on mesoscopic dynamic models, these parameters have been determined with data-fitting techniques. In this study, we describe structural dynamics in terms of the eigenmodes comprising the SRLS time correlation functions (TCFs) generated by using the best-fit parameters as input to the Smoluchowski equation. An eigenmode is a weighted exponential with decay constant given by an eigenvalue of the Smoluchowski operator, and weighting factor determined by the corresponding eigenvector. Obviously, both quantities depend on the SRLS parameters as determined by the SRLS model. Unlike the set of best-fit parameters, the eigenmodes represent patterns of motion of the probe-protein system. The following new information is obtained for the typical probe, the (15)N-(1)H bond. Two eigenmodes, associated with the protein and the probe, dominate when the time scale separation is large (i.e., D2 >> D1), the tensorial properties are simple, and the local potential is either very strong or very weak. When the potential exceeds these limits while the remaining conditions are preserved, new eigenmodes arise. The multi-exponentiality of the TCFs is associated in this case with the restricted nature of the local motion. When the time scale separation is no longer large, the rotational degrees of freedom of the protein and the probe become statistically dependent (coupled dynamically). The multi-exponentiality of the TCFs is associated in this case with the restricted nature of both the local and the global motion. The effects of local diffusion axiality, potential strength, and extent of mode-coupling on the eigenmode setup are investigated. We detect largely global motional or largely local motional eigenmodes. In addition, we detect mixed eigenmodes associated with correlated∕prograde or anti-correlated∕retrograde rotations of the global (D1) and local (D2) motional modes. The eigenmode paradigm is applied to N-H bond dynamics in the β-sheet residue K19, and the α-helix residue A34, of the third immunoglobulin-binding domain of streptococcal protein G. The largest contribution to the SRLS TCFs is made by mixed anti-correlated D1 and D2 eigenmodes. The next largest contribution is made by D1-dominated eigenmodes. Eigenmodes dominated by the local motion contribute appreciably to A34 and marginally to K19. Correlated D1 and D2 eigenmodes contribute exclusively to K19 and do not contribute above 1% to A34. The differences between K19 and A34 are delineated and rationalized in terms of the best-fit SRLS parameters and mode-mixing. It may be concluded that eigenmode analysis is complementary and supplementary to data-fitting-based analysis.

Starting‐structure dependence of nanosecond timescale intersubstate transitions and reproducibility of MD‐derived order parameters

Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin-binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A, J Am Chem Soc 2010;132:4295-4309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR-derived order parameters. Thus, MD-derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions.

Worm-Like Ising Model for Protein Mechanical Unfolding under the Effect of Osmolytes

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

The temperature intervals with global exchange of replicas empirical accelerated sampling method: Parameter sensitivity and extension to a complex molecular system

The recently developed "temperature intervals with global exchange of replicas" (TIGER2) algorithm is an efficient replica-exchange sampling algorithm that provides the freedom to specify the number of replicas and temperature levels independently of the size of the system and temperature range to be spanned, thus making it particularly well suited for sampling molecular systems that are considered to be too large to be sampled using conventional replica exchange methods. Although the TIGER2 method is empirical in nature, when appropriately applied it is able to provide sampling that satisfies the balance condition and closely approximates a Boltzmann-weighted ensemble of states. In this work, we evaluated the influence of factors such as temperature range, temperature spacing, replica number, and sampling cycle design on the accuracy of a TIGER2 simulation based on molecular dynamics simulations of alanine dipeptide in implicit solvent. The influence of these factors is further examined by calculating the properties of a complex system composed of the B1 immunoglobulin-binding domain of streptococcal protein G (protein G) in aqueous solution. The accuracy of a TIGER2 simulation is particularly sensitive to the maximum temperature level selected for the simulation. A method to determine the appropriate maximum temperature level to be used in a TIGER2 simulation is presented.

Structural analysis of protein dynamics by MD simulations and NMR spin‐relaxation

Molecular dynamics (MD) simulations and nuclear magnetic resonance spin-relaxation measurements provide detailed insights into ps-ns structural dynamics of proteins. An analysis of discrepancies between the two methods is presented for the B3 immunoglobulin-binding domain of streptococcal protein G. MD simulations using three MD force fields (OPLS-AA, AMBER ff99SB, and AMBER ff03) overestimate the flexibility of backbone N--H vectors at the borders of secondary structure and in loops when compared with experimentally determined backbone amide generalized order parameters (Hall and Fushman, J Am Chem Soc 2006; 12:7855-7870). Comparison with a previous study of residual dipolar coupling constants (Bouvignies et al., Proc Natl Acad Sci USA 2005;102:13885-13890) indicates that slower timescale motions do not account for the discrepancies. Structural analysis reveals that relative imbalance between the description of hydrogen bonding and other terms of modern force fields may be responsible for disagreement.

13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy

Complete 13C and 15N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state shifts.

Long dynamics simulations of proteins using atomistic force fields and a continuum representation of solvent effects: calculation of structural and dynamic properties.

Long dynamics simulations were carried out on the B1 immunoglobulin-binding domain of streptococcal protein G (ProtG) and bovine pancreatic trypsin inhibitor (BPTI) using atomistic descriptions of the proteins and a continuum representation of solvent effects. To mimic frictional and random collision effects, Langevin dynamics (LD) were used. The main goal of the calculations was to explore the stability of tens-of-nanosecond trajectories as generated by this molecular mechanics approximation and to analyze in detail structural and dynamical properties. Conformational fluctuations, order parameters, cross correlation matrices, residue solvent accessibilities, pKa values of titratable groups, and hydrogen-bonding (HB) patterns were calculated from all of the trajectories and compared with available experimental data. The simulations comprised over 40 ns per trajectory for ProtG and over 30 ns per trajectory for BPTI. For comparison, explicit water molecular dynamics simulations (EW/MD) of 3 ns and 4 ns, respectively, were also carried out. Two continuum simulations were performed on each protein using the CHARMM program, one with the all-atom PAR22 representation of the protein force field (here referred to as PAR22/LD simulations) and the other with the modifications introduced by the recently developed CMAP potential (CMAP/LD simulations). The explicit solvent simulations were performed with PAR22 only. Solvent effects are described by a continuum model based on screened Coulomb potentials (SCP) reported earlier, i.e., the SCP-based implicit solvent model (SCP-ISM). For ProtG, both the PAR22/LD and the CMAP/LD 40-ns trajectories were stable, yielding C(alpha) root mean square deviations (RMSD) of about 1.0 and 0.8 A respectively along the entire simulation time, compared to 0.8 A for the EW/MD simulation. For BPTI, only the CMAP/LD trajectory was stable for the entire 30-ns simulation, with a C(alpha) RMSD of approximately 1.4 A, while the PAR22/LD trajectory became unstable early in the simulation, reaching a C(alpha) RMSD of about 2.7 A and remaining at this value until the end of the simulation; the C(alpha) RMSD of the EW/MD simulation was about 1.5 A. The source of the instabilities of the BPTI trajectories in the PAR22/LD simulations was explored by an analysis of the backbone torsion angles. To further validate the findings from this analysis of BPTI, a 35-ns SCP-ISM simulation of Ubiquitin (Ubq) was carried out. For this protein, the CMAP/LD simulation was stable for the entire simulation time (C(alpha) RMSD of approximately 1.0 A), while the PAR22/LD trajectory showed a trend similar to that in BPTI, reaching a C(alpha) RMSD of approximately 1.5 A at 7 ns. All the calculated properties were found to be in agreement with the corresponding experimental values, although local deviations were also observed. HB patterns were also well reproduced by all the continuum solvent simulations with the exception of solvent-exposed side chain-side chain (sc-sc) HB in ProtG, where several of the HB interactions observed in the crystal structure and in the EW/MD simulation were lost. The overall analysis reported in this work suggests that the combination of an atomistic representation of a protein with a CMAP/CHARMM force field and a continuum representation of solvent effects such as the SCP-ISM provides a good description of structural and dynamic properties obtained from long computer simulations. Although the SCP-ISM simulations (CMAP/LD) reported here were shown to be stable and the properties well reproduced, further refinement is needed to attain a level of accuracy suitable for more challenging biological applications, particularly the study of protein-protein interactions.

Protein modeling with reduced representation: statistical potentials and protein folding mechanism.

A high resolution reduced model of proteins is used in Monte Carlo dynamics studies of the folding mechanism of a small globular protein, the B1 immunoglobulin-binding domain of streptococcal protein G. It is shown that in order to reproduce the physics of the folding transition, the united atom based model requires a set of knowledge-based potentials mimicking the short-range conformational propensities and protein-like chain stiffness, a model of directional and cooperative hydrogen bonds, and properly designed knowledge-based potentials of the long-range interactions between the side groups. The folding of the model protein is cooperative and very fast. In a single trajectory, a number of folding/unfolding cycles were observed. Typically, the folding process is initiated by assembly of a native-like structure of the C-terminal hairpin. In the next stage the rest of the four-ribbon beta-sheet folds. The slowest step of this pathway is the assembly of the central helix on the scaffold of the beta-sheet.

A simple method to measure 13CH 2 heteronuclear dipolar cross-correlation spectral densities

Here, we report a method to simultaneously determine CH 2 cross-correlation spectral densities and T 1 relaxation times in the laboratory and rotating frames. To accomplish this, we have employed an indirect approach that is based on measurement of differences in relaxation rates acquired with and without cross-correlation terms. The new method, which can be employed using multidimensional NMR and standard relaxation pulse sequences, is validated experimentally by investigation of a selectively 13C-enriched hexadecapeptide and the uniformly 13C-enriched immunoglobulin-binding domain of streptococcal protein G (GB1). Use of this approach makes determination of CH 2 cross-correlation spectral densities in uniformly 13C-enriched proteins now routine and provides novel information concerning their internal motions.

Comparison of (13)C(alpha)H and (15)NH backbone dynamics in protein GB1.

This study presents a site-resolved experimental view of backbone C(alpha)H and NH internal motions in the 56-residue immunoglobulin-binding domain of streptococcal protein G, GB1. Using (13)C(alpha)H and (15)NH NMR relaxation data [T(1), T(2), and NOE] acquired at three resonance frequencies ((1)H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(omega) = 2omegaJ(omega) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(omega) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C(alpha)H and NH groups. Deconvolution of F(omega) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C(alpha)H internal motions are more broadly distributed on the nanosecond time scale, and larger C(alpha)H order parameters are related to correlated bond rotations for C(alpha)H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of beta-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.

A rapid method to attain isotope labeled small soluble peptides for NMR studies.

A widely applicable strategy is presented for efficient and rapid production of small water soluble peptides expressed as fusion proteins with the immunoglobulin-binding domain of streptococcal protein G. A simple extraction and purification scheme that includes a protease cleavage step to release the target peptide is described. The yield of authentic target peptide exceeds 10 mg per liter of culture. Production of U-13C, 15N and highly deuterated U-13C, 15N isotope labeled peptide is demonstrated for the 11 residue S2 peptide, corresponding to the C-terminus of the alpha-subunit of transducin, and the coiled coil trimerization domain from cartilage matrix protein (CMPcc), respectively. Heteronuclear two-dimensional NMR spectra are used for initial peptide characterization.

Heat Capacities and a Snapshot of the Energy Landscape in Protein GB1 from the Pre-denaturation Temperature Dependence of Backbone NH Nanosecond Fluctuations

Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone 15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5–50 °C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the F(ω)=2ω J(ω) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33 kJ/mol residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most “rigid” segments of the fold: β-strands 1 and 4 and the α-helix. Trends in this landscape also parallel the free energy of folding–unfolding derived from hydrogen–deuterium (H–D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H–D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100 J/mol K residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.

Role of side-chains in the cooperative beta-hairpin folding of the short C-terminal fragment derived from streptococcal protein G.

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR.

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.