Dynamics and thermodynamics of the regulatory domain of human cardiac troponin C in the apo- and calcium-saturated states

The contraction of cardiac and skeletal muscles is triggered by the binding of Ca2+ to their respective troponin C (TnC) proteins. Recent structural data of both cardiac and skeletal TnC in both the apo and Ca2+ states have revealed that the response to Ca2+ is fundamentally different for these two proteins. For skeletal TnC, binding of two Ca2+ to sites 1 and 2 leads to large changes in the structure, resulting in the exposure of a hydrophobic surface. For cardiac TnC, Ca2+ binds site 2 only, as site 1 is inactive, and the structures show that the Ca2+-induced changes are much smaller and do not result in the exposure of a large hydrophobic surface. To understand the differences between regulation of skeletal and cardiac muscle, we have investigated the effect of Ca2+ binding on the dynamics and thermodynamics of the regulatory N-domain of cardiac TnC (cNTnC) using backbone 15N nuclear magnetic resonance relaxation measurements for comparison to the skeletal system. Analysis of the relaxation data allows for the estimation of the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy of the Ca2+-binding sites on a per residue basis, which can be related to the structural features of the sites. The results indicate that binding of Ca2+ to the functional site in cNTnC makes the site more rigid with respect to high-frequency motions; this corresponds to a decrease in the conformational entropy (TdeltaS) of the site by 2.2 kcal mol(-1). Although site 1 is defunct, binding to site 2 also decreases the conformational entropy in the nonfunctional site by 0.5 kcal mol(-1). The results indicate that the Ca2+-binding sites in the regulatory domain are structurally and energetically coupled despite the inability of site 1 to bind Ca2+. Comparison between the cardiac and skeletal isoforms in the apo state shows that there is a decrease in conformational entropy of 0.9 kcal mol(-1) for site 1 of cNTnC and little difference for site 2.     

1H-NMR resonance assignments, secondary structure, and global fold of the TR1C fragment of turkey skeletal troponin C in the calcium-free state

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].     

Application of a self-consistent mean field theory to predict protein side-chains conformation and estimate their conformational entropy

Understanding the relations between the conformation of the side-chains and the backbone geometry is crucial for structure prediction as well as for homology modelling. To attempt to unravel these rules, we have developed a method which allows us to predict the position of the side-chains from the co-ordinates of the main-chain atoms. This method is based on a rotamer library and refines iteratively a conformational matrix of the side-chains of a protein, CM, such that its current element at each cycle CM (ij) gives the probability that side-chain i of the protein adopts the conformation of its possible rotamer j. Each residue feels the average of all possible environments, weighted by their respective probabilities. The method converges in only a few cycles, thereby deserving the name of self consistent mean field method. Using the rotamer with the highest probability in the optimized conformational matrix to define the conformation of the side-chain leads to the result that on average 72% of chi 1, 75% of chi 2 and 62% of chi 1 + 2 are correctly predicted for a set of 30 proteins. Tests with six pairs of homologous proteins have shown that the method is quite successful even when the protein backbone deviates from the correct conformation. The second application of the optimized conformational matrix was to provide estimates of the conformational entropy of the side-chains in the folded state of the protein. The relevance of this entropy is discussed.     

Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution

We have solved and refined the crystal and molecular structures of the calcium-saturated N-terminal domain of troponin C (TnC) to 1.75 A resolution. This has allowed for the first detailed analysis of the calcium binding sites of this molecular switch in the calcium-loaded state. The results provide support for the proposed binding order and qualitatively, for the affinity of calcium in the two regulatory calcium binding sites. Based on a comparison with the high-resolution apo-form of TnC we propose a possible mechanism for the calcium-mediated exposure of a large hydrophobic surface that is central to the initiation of muscle contraction within the cell.     

Interactions between domains of apo calmodulin alter calcium binding and stability

Calmodulin (CaM) is an essential protein that exerts exquisite spatial and temporal control over diverse eukaryotic processes. Although the two half-molecule domains of CaM each have two EF-hands and bind two calcium ions cooperatively, they have distinct roles in activation of some targets. Interdomain interactions may mediate coordination of their actions. Proteolytic footprinting titrations of CaM [Pedigo and Shea (1995) Biochemistry 34, 1179-1196; Shea, Verhoeven, and Pedigo (1996) Biochemistry 35, 2943-2957] showed that calcium binding to the high-affinity sites (III and IV in the C-domain) alters the conformation of helix B in the N-domain despite sites I and II being vacant. This may arise from calcium-induced disruption of interactions between the apo domains. In this study, comparing the cloned domains (residues 1-75, 76-148) to whole CaM, the proteolytic susceptibility of helix B in the apo isolated N-domain was higher than in apo CaM. The isolated N-domain was monotonically protected by calcium binding and had a higher calcium affinity than when part of whole CaM. The change in affinity was small (1-1.5 kcal/mol) but acted to separate the domain saturation curves of whole CaM. Unfolding enthalpies and melting temperatures of the apo isolated domains did not correspond to the two transitions resolved for apo CaM. In summary, the interactions between domains of apo CaM protected the N-domain from proteolysis and raised its Tm by 10 degrees C, demonstrating that CaM is not the sum of its parts.     

Using a feminist framework for investigating staff nurses'' philosophies of nursing

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca2+ concentration. The binding of Ca2+ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (tau r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg2+ by Ca2+ at these sites (tau r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca2+ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl.     

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.     

Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site

The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Calpha, C', HN, Halpha, and side chain Cbeta resonance assignments. The remaining 1H, 13C, and 15N resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene Hbeta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by 15N T1, T2, and heteronuclear 15N-1H NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time taum of 4.88 +/- 0.04 ns and provide evidence for fast time scale (taue < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.     

Dynamics study on the anti-human immunodeficiency virus chemokine viral macrophage-inflammatory protein-II (VMIP-II) reveals a fully monomeric protein

Encoded by Kaposi's sarcoma-associated herpesvirus, viral macrophage-inflammatory protein-II (VMIP-II) is unique among CC chemokines in that it has been shown to bind to the CXC chemokine receptor CXCR4 as well as to a variety of CC chemokine receptors. This unique binding ability allows vMIP-II to block infection by a wide range of human immunodeficiency virus type I (HIV-1) strains, but the structural and dynamic basis for this broad range of binding is not known. 15N T1, T2 and 15N[-HN] nuclear Overhauser effect (NOE) values of vMIP-II, determined through a series of heteronuclear multidimensional nuclear magnetic resonance (NMR) experiments, were used to obtain information about the backbone dynamics of the protein. Whereas almost all chemokine structures reveal a dimer or multimer, vMIP-II has a rotational correlation time (tauc) of 4.7 +/- 0.3 ns, which is consistent with a monomeric chemokine. The rotational diffusion anisotropy, D parallel/D perpendicular, is approximately 1.5 +/- 0.1. The conformation of vMIP-II is quite similar to other known chemokines, containing an unstructured N-terminus followed by an ordered turn, three beta-strands arranged in an antiparallel fashion, and one C-terminal alpha-helix that lies across the beta-strands. Most of the protein is well-ordered on a picosecond time scale, with an average order parameter S2 (excluding the N-terminal 13 amino acids) of 0.83 +/- 0. 09, and with even greater order in regions of secondary structure. The NMR data reveal that the N-terminus, which in other chemokines has been implicated in receptor binding, extends like a flexible tail in solution and possesses no secondary structure. The region of the ordered turn, including residues 25-28, experiences conformational exchange dynamics. The implications of these NMR data to the broad receptor binding capability of vMIP-II are discussed.     

Characterization of lanthanide ion binding to the EF-hand protein S100 beta by luminescence spectroscopy

S100 beta is a member of a group of low-molecular weight acidic calcium binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and II). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy using Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characterize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta shows two distinct Eu3+ binding environments from both the excitation spectrum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd of 660 +/- 20 nM, whereas the dissociation constant for the pseudo-EF hand site I is significantly weaker. Lifetimes in H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water molecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln3+ binding domains I and II in calmodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta show that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is significantly different than that in the absence of Zn2+. Tyrosine-17-sensitized Tb3+ luminescence experiments indicate that the Tb3+ occupying the proximal weaker binding site I is sensitized, whereas Tb3+ in site II is not. The distance between sites I and II (15.0 +/- 0.4 A) in S100 beta was determined from Forster-type energy transfer in D2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Zn2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 A. Location of histidine-15 close to pseudo-EF site I suggests that Zn2+ binding likely changes the conformation of this site, causing a reduction of the intersite distance by approximately 2 A.     

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.     

Relationship between enzyme specificity and the backbone dynamics of free and inhibited alpha-lytic protease

To better understand the structural basis for the observed patterns in substrate specificity, the backbone dynamics of alpha-lytic protease have been investigated using 15N relaxation measurements. The enzyme was inhibited with the peptide boronic acid N-tert-butyloxycarbonyl-Ala-Pro-boroVal [Kettner, C. A., et al. (1988) Biochemistry 27, 7682], which mimics interactions occurring in the tetrahedral transition state or nearby intermediates, and the dynamics of the unbound and inhibited enzyme were compared. Arrayed 2-D NMR spectra were acquired to measure T1, T2, and steady-state ?1H?-15N NOE of >95% of the backbone amides in both protein samples. The overall rotational correlation time tauc was found to be 8.1 ns. Values of the spectral density function J(omega) at omega = 0, omegaN, and approximately omegaH were derived from the relaxation results using reduced spectral density mapping [Ishima, R., & Nagayama, K. (1995) Biochemistry 34, 3162]. The resultant spectral densities were interpreted to indicate regions of fast motion (nanosecond to picosecond) and of intermediate chemical exchange (millisecond to microsecond). The protein has 13 regions with increased motion on the fast time scale; these generally fall on exterior turns and loops and most correlate with regions of higher crystallographic B-factors. Several stretches of backbone undergo intermediate chemical exchange, indicating motion or other processes that cause temporal chemical shift changes. A comparison of spectral densities for both the free and inhibited enzymes revealed that inhibitor binding preferentially stabilizes regions undergoing chemical exchange (which predominate around the active site) and only minimally affect regions of rapid motion. Slow motions, suggestive of backbone plasticity, are observed in most of the binding pocket residues. This may point to a mechanism for the observed broad specificity of the enzyme. The significance of the observed dynamics for substrate binding and specificity is discussed.     

Thermodynamics of a reverse turn motif. Solvent effects and side-chain packing

The linear pentapeptide, Ala-Tyr-cis-Pro-Tyr-Asp-NMA (AYPYD) is known to have a significant population of type VI turn conformers in aqueous solvent. We have carried out theoretical studies of the conformational energetics of this peptide using a potential of mean force (PMF) consisting of the AMBER/OPLS empirical potential energy function, a macroscopic electrostatic model of polar solvation, and a surface area-based model of non-polar solvation. Conformers were taken from molecular dynamics simulations reported elsewhere, or generated by a random search method reported here. The chain entropy of folding was calculated by a systematic search of accessible dihedral angle space. The intra-peptide component was found to strongly favor folding and was nearly cancelled by the polar solvation term which disfavored folding. The non-polar solvation term had little effect. Fluctuations about the average value of the PMF were small and in accord with estimates from a simple harmonic model. When applied to conformers generated by a random search, the PMF selected a conformer close to the NMR-determined structure as the lowest energy conformer. The conformer with the second-lowest energy was extended, but was found to fold rapidly to the turn state in a subsequent molecular dynamics study, and may be an important state on the folding-unfolding pathway. Averages of the PMF were combined with the entropy estimates to provide an estimate of the free energy of folding that is in reasonable agreement with experimental results. In terms of the interplay between backbone electrostatic interactions and the packing of apolar side-chains, this peptide provides a model for the energetics of protein folding, and therefore makes a useful test case for calculations.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.     

Molecular basis of resistance to HIV-1 protease inhibition: a plausible hypothesis

The binding thermodynamics of the HIV-1 protease inhibitor acetyl pepstatin and the substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln, corresponding to one of the cleavage sites in the gag, gag-pol polyproteins, have been measured by direct microcalorimetric analysis. The results indicate that the binding of the peptide substrate or peptide inhibitor is entropically driven; i.e., it is characterized by an unfavorable enthalpy and a favorable entropy change, in agreement with a structure-based thermodynamic analysis based upon an empirical parameterization of the energetics. Dissection of the binding enthalpy indicates that the intrinsic interactions are favorable and that the unfavorable enthalpy originates from the energy cost of rearranging the flap region in the protease molecule. In addition, the binding is coupled to a negative heat capacity change. The dominant binding force is the increase in solvent entropy that accompanies the burial of a significant hydrophobic surface. Comparison of the binding energetics obtained for the substrate with that obtained for synthetic nonpeptide inhibitors indicates that the major difference is in the magnitude of the conformational entropy change. In solution, the peptide substrate has a higher flexibility than the synthetic inhibitors and therefore suffers a higher conformational entropy loss upon binding. This higher entropy loss accounts for the lower binding affinity of the substrate. On the other hand, due to its higher flexibility, the peptide substrate is more amenable to adapt to backbone rearrangements or subtle conformational changes induced by mutations in the protease. The synthetic inhibitors are less flexible, and their capacity to adapt is more restricted. The expected result is a more pronounced effect of mutations on the binding affinity of the synthetic inhibitors. On the basis of the thermodynamic differences in the mode of binding of substrate and synthetic inhibitors, it appears that a key factor to understanding resistance is given by the relative balance of the different forces that contribute to the binding free energy and, in particular, the balance between conformational and solvation entropy.     

Consistency in structural energetics of protein folding and peptide recognition

We report a new free energy decomposition that includes structure-derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single-domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single-domain proteins for which the three-dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (delta Sbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence-dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA-A2, 1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.     

Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.     

Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.