We have used differential scanning calorimetry to determine the effect of low concentrations (C = 0–2 M) of the osmolyte sarcosine on the Gibbs energy changes (ΔG) for the unfolding of hen-egg-white lysozyme, ribonuclease A, and ubiquitin, under the same buffer and pH conditions. We have also computed this effect on the basis of the additivity assumption and using published values of the transfer Gibbs energies for the amino acid side chains and the peptide backbone unit. The values thus predicted for the slope ∂ΔG/∂C agree with the experimental ones, but only if the unfolded state is assumed to be compact (that is, if the accessibility to solvent of the unfolded state is modeled using segments excised from native structures). The additivity-based calculations predict similar ∂ΔG/∂C values for the three proteins studied. We point out that, to the extent that this approximate constancy of ∂ΔG/∂C holds, osmolyte-induced increases in denaturation temperature will be larger for proteins with low unfolding enthalpy (small proteins that bury a large proportion of apolar surface). The experimental results reported here are consistent with this hypothesis.

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 105-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 °C results in a 103-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 °C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer–dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 °C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of “monomeric” proteins could be more prevalent than is usually appreciated.

Analysis of thermal melting curves represents one important approach for evaluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differential melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (Tm) and van't Hoff enthalpy (ΔHvH). Calculated Tm and ΔHvH values are insensitive to the choice of pre- and post-transition baselines. Consequently, the method accurately computes Tm and ΔHvH for extremely truncated data sets, in the complete absence of baseline information, and for proteins with low melting temperatures, where the traditional direct approach routinely fails. Moreover, agreement between ΔHvH values obtained using points derived from pre- vs. post-transition data provide an independent method for detecting some classes of non-two-state transitions. Finally, fitting of the differential denaturation curve should prove useful for analysis of abbreviated data sets obtained from high throughput array analysis of protein stability.

A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (ΔGu), melting temperatures (Tm), and cooperativity. Metropolis-driven simulated annealing and low-temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gβ1, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side-chain conformations remarkably similar to that of native were predicted.

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.

The Escherichia coli trigger factor is a peptidyl–prolyl cis–trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced αLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd ∼ 70–90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin–target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.

The wrong Figure 2 was used in the above article that appeared in Volume 8, Number 11, pages 2223–2233. We are reproducing the correct Figure 2 here.

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2 . The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H–1H and 1H–15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen–deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.

When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all-atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all-atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active-site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild-type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.

The standard molecular model of the fiber of the sickle hemoglobin (HbS: β6 Glu → Val) has been revised to allow both β6 mutation sites to participate in intermolecular contacts, rather than only one β6 site as previously thought, for four molecules per 14-molecule fiber cross section. This structure accurately predicts the copolymerization of hybridized mixtures of HbS with HbA or HbC (β6 Glu → Lys), which could not be reconciled with prior models in which only half the β6 sites were required for assembly. This model suggests new contacts within the fiber and raises the question of whether these cross-linked double strands could possess added stability important in such processes as nucleation.

The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate d-dehydrogenase in a ternary complex with NADPH and l-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 Å. l-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 μM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+-dependent oxidation of the d-amino acid stereocenter of meso-diaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a d-amino acid. The enzyme is involved in the biosynthesis of meso-diaminopimelate and l-lysine from l-aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated l-2-amino-6-methylene-pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso-diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the α-l-amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Iα) of hen lysozyme with α-domain folded and β-domain unfolded is on the folding pathway under the classical definition. Iα folds to the native (N) state directly (unfolded (U) [hArr ] Iα [hArr ] N) without having to unfold to U and then refold to N through alternative folding pathways as in Iα [hArr ] U [hArr ] N.

α-Synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of α-synuclein has not yet been established. We here present biochemical evidence that recombinant α-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, α-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and α-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of α-synuclein, which is believed to reorganize the molecular surface of α-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, α-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, α-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that α-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that α-synuclein could act as a molecular chaperone, and that the conformational change of the α-synuclein could explain the aggregation kinetics of α-synuclein, which may be related to the abolishment of the chaperonic-like activity.

In the early morning of January 11, 2000, while walking from home to his office at the J. Willard Gibbs Laboratory, Paul Sigler, one of the giants in structural biology of our time, collapsed and passed away. While 65 years old, an age when many may be resting on past successes, Paul was in mid-course, achieving greater and greater heights of structural and chemical understanding of basic biological systems of transcription, signal transduction, interfacial catalysis, and chaperonin-mediated protein folding. As a father of five grown children, he was also savoring, with his wife Jo, the arrival and maturation of eight grandchildren living in the United States, England, and Israel. We extend our deepest sympathies to his family.

Escherichia coli strain LE316 contains a mutation in gyrB that results in the substitution of Val164 to Gly and confers both chlorobiocin resistance and temperature sensitivity. Selection for suppressors of the ts phenotype yielded second-site mutations in GyrB at His38 and Thr157. The properties of proteins bearing these mutations have been characterized, and a mechanism of suppression is proposed based upon structural considerations.

α1-Antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of α1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that α1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the β-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a β-strand linked polymer form of α1-antitrypsin: the crystal structure of a cleaved α1-antitrypsin polymer.

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623–644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361–369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic l-Asp/l-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of l-Asp by l-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (l-Asp/l-isoAsp)-X bond and of the X-(l-Asp/l-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either l-Asp or l-isoAsp, these identify one or the other form. Characterization and relative quantification of l-Asp/l-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1–7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41–46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single “acid form” (A), and a homogeneous “basic form” (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, “closed” conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.