1H n.m.r. studies of insulin. Assignment of resonances and properties of tyrosine residues.

The assignment of the aromatic 1H n.m.r. resonances of the four tyrosine residues of bovine 2-zinc insulin is reported, based on double resonance techniques, use of Hahn spin echo pulse sequences and examination of specific derivatives nitrated at tyrosines A14 and A19 as well as des-(B26-B30)-insulin. Titration curves of the four tyrosine residues show that residues A14 and B16 have normal pK' values of 10.3-10.6 in solution, consistent with their accessibility to solvent in monomer and dimer in the crystal. Tyrosine residues A19 and B26 have pK' values of 11.4 and exhibit other features in their titration curves that are consistent with limited accessibility to solvent and a nonpolar environment. The meta protons of residues B16 and B26 both observe the titration of a nearby tyrosine residue, probably A19. Interpretation of the n.m.r. data obtained in solution is consistent with the crystallographic data for the monomer and dimer obtained on insulin crystals [Blundell, Dodson, Hodgkin & Mercola (1972) Adv. Protein Chem. 26, 279-402].     

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.     

Novel inter-protein cross-link identified in the GGH-ecotin D137Y dimer

In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.     

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.     

Stoichiometric arginine binding in the oxygenase domain of inducible nitric oxide synthase requires a single molecule of tetrahydrobiopterin per dimer

In addition to its catalytic roles, the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (H4B) is required for substrate binding and for stabilization of the dimeric structure. We expressed and purified the core of the iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the presence (H4B+) and absence (H4B-) of this cofactor. Both forms bound stoichiometric amounts of heme (>0.9 heme per protein subunit). H4B- CODiNOS was unable to bind arginine, gave an unstable ferrous carbonyl adduct, and was a mixture of monomer and dimer. H4B+ CODiNOS bound arginine, gave a stable ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichiometric titrations.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Dimeric form of diphtheria toxin: purification and characterization

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.     

Structure of the tubulin dimer

Microtubules are formed from a 110,000-dalton dimeric subunit called tubulin. Two forms of 55,000-dalton monomer, alpha and beta, are found in all microtubule preparations. The dimers could thus theoretically be either heterodimers (alphabeta) or homodimers (alphaalpha and betabeta). This problem was investigated by stigated by chemical cross-linking using several bifunctional reagents, of which one, dimethyl-3,3-(tetrame thylenedioxy) dipropionimidate dihydrochloride (DTDI), was able to make intradimer bonds in tubulin. When soluble chick brain tubulin was cross-linked with DTDI and analyzed by electrophoresis in an acrylamide gel system capable of resolving alphaalpha, alphabeta, and betabeta, 60 to 90% of the cross-linked dimer was alphabeta. If tubulin was incubated at 24 degrees prior to cross-linking with DTDI the total yield of cross-linked dimer increased with time, indicating that tubulin was forming loose aggregates. The relative amounts of cross-linked dimer alphaalpha and betabeta also increase with time, indicating that soluble tubulin is largely alphabeta, and suggesting that cross-linked alphaalpha and betabeta arise from nonspecific aggregation during tubulin purification. The aggregation observed by cross-linking with DTDI was strongly influenced by colchicine and Vinca alkaloids in a pattern similar to the effects of these drugs on tubulin polymerization.     

Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.     

Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

The effect of iodination of particular tyrosine residues on the hormonal activity of insulin

Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The resultant preparations were fragmented in various ways and the ratio of tyrosine to monoiodotyrosine and di-iodotyrosine was determined in each fragment. This has allowed the distribution of iodine between the combined A-chain tyrosine residues and the individual B-chain tyrosine residues to be determined. The hormonal activity of each of these iodinated insulin preparations was measured from their effect on the production of (14)CO(2) from [1-(14)C]glucose by isolated adipose cells. The results were interpreted as meaning that the iodination of tyrosine residue A19 or B16 leads to the inactivation of insulin. Speculations are made about the nature of an interaction between insulin and a receptor site on the target tissue.     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.     

Solution structure of the disulfide-linked dimer of human intestinal trefoil factor (TFF3): the intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins

The trefoil protein TFF3 forms a homodimer (via a disulfide linkage) that is thought to have increased biological activity over the monomer. The solution structure of the TFF3 dimer has been determined by NMR and compared with the structure of the TFF3 monomer and with other trefoil dimer structures (TFF1 and TFF2). The most significant structural differences between the trefoil domain in the monomer and dimer TFF3 are in the orientations of the N-terminal 3(10)-helix (residues 10-12) and in the presence in the dimer of an additional 3(10)-helix (residues 53-55) outside of the core region. The TFF3 dimer forms a more compact structure as compared with the TFF1 dimer where the two trefoil domains are connected by a flexible region with the monomer units being at variable distances from each other and in many different orientations. Although TFF2 is also a compact structure, the dispositions of its monomer units are very different from those of TFF3. The structural differences between the dimers result in the two putative receptor/ligand binding sites that remain solvent exposed in the dimeric structures having very different dispositions in the different dimers. Such differences have significant implications for the mechanism of action and functional specificity for the TFF class of proteins.     

1H n.m.r. studies of insulin. Reversible transformation of 2-zinc to 4-zinc insulin hexamer

1H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-Bl-insulin did not undergo the transformation. Addition of SCN- to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn2+ promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.