Subharmonic lock-in detection and its optimization for femtosecond noise correlation spectroscopy.

Although often viewed as detrimental, fluctuations carry valuable information about the physical system from which they emerge. Femtosecond noise correlation spectroscopy (FemNoC) has recently been established to probe the ultrafast fluctuation dynamics of thermally populated magnons by measurement of their amplitude autocorrelation. Subharmonic lock-in detection is the key technique in this method, allowing us to extract the pulse-to-pulse polarization fluctuations of two femtosecond optical pulse trains transmitted through a magnetic sample. Here, we present a thorough technical description of the subharmonic demodulation technique and the FemNoC measurement system. We mathematically model the data acquisition process and identify the essential parameters that critically influence the signal-to-noise ratio of the signals. Comparing the model calculations to real datasets allows validating the predicted parameter dependences and provides a means to optimize FemNoC experiments.

Discovery of ultrafast spontaneous spin switching in an antiferromagnet by femtosecond noise correlation spectroscopy.

Owing to their high magnon frequencies, antiferromagnets are key materials for future high-speed spintronics. Picosecond switching of antiferromagnetic spin systems has been viewed a milestone for decades and pursued only by using ultrafast external perturbations. Here, we show that picosecond spin switching occurs spontaneously due to thermal fluctuations in the antiferromagnetic orthoferrite Sm0.7Er0.3FeO3. By analysing the correlation between the pulse-to-pulse polarisation fluctuations of two femtosecond optical probes, we extract the autocorrelation of incoherent magnon fluctuations. We observe a strong enhancement of the magnon fluctuation amplitude and the coherence time around the critical temperature of the spin reorientation transition. The spectrum shows two distinct features, one corresponding to the quasi-ferromagnetic mode and another one which has not been previously reported in pump-probe experiments. Comparison to a stochastic spin dynamics simulation reveals this new mode as smoking gun of ultrafast spontaneous spin switching within the double-well anisotropy potential.

Design of non-standard insulin analogs for the treatment of diabetes mellitus.

Structure-based protein design has enabled the engineering of insulin analogs with improved pharmacokinetic and pharmacodynamic properties. Exploiting classical structures of zinc insulin hexamers, the first insulin analog products focused on destabilization of subunit interfaces to obtain rapid-acting (prandial) formulations. Complementary efforts sought to stabilize the insulin hexamer or promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus; Sanofi-Aventis, Paris, France) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir; Novo-Nordisk, Basværd, Denmark). In the past year second-generation basal insulin analogs have entered clinical trials in an effort to obtain ideal flat 24-hour pharmacodynamic profiles. The strategies employ non-standard protein modifications. One candidate (insulin degludec; Novo-Nordisk a/s) undergoes extensive subcutaneous supramolecular assembly coupled to a large-scale allosteric reorganization of the insulin hexamer (the TR transition). Another candidate (LY2605541; Eli Lilly and Co., Indianapolis, IN, USA) utilizes coupling to polyethylene glycol to delay absorption and clearance. On the other end of the spectrum, advances in delivery technologies (such as microneedles and micropatches) and excipients (such as the citrate/zinc-ion chelator combination employed by Biodel, Inc., Danbury, CT, USA) suggest strategies to accelerate PK/PD toward ultra-rapid-acting insulin formulations. Next-generation insulin analogs may also address the feasibility of hepatoselective signaling. Although not in clinical trials, early-stage technologies provide a long-range vision of "smart insulins" and glucose-responsive polymers for regulated hormone release.

Varying ratios of wavelengths in dual wavelength LED photomodulation alters gene expression profiles in human skin fibroblasts.

BACKGROUND AND OBJECTIVE: LED photomodulation has been shown to profoundly influence cellular behavior. A variety of parameters with LED photomodulation can alter cellular response in vitro. The effects of one visible and one infrared wavelength were evaluated to determine the optimal ratio to produce a net increase in dermal collagen by altering the ratio of total energy output of each wavelength. The ratio between the two wavelengths (590 and 870 nm) was shifted in 25% increments. STUDY DESIGN/MATERIALS AND METHODS: Human skin fibroblasts in culture were exposed to a 590/870 nm LED array with total combined energy density fixed at 4.0 mW/cm.. The ratio of 590/870 nm tested parameters were: 100/0%, 75/25%, 50/50%, 25/75%, and 0/100%. These ratios were delivered using pulsed duty cycle of exposure (250 milliseconds "on" time/100 milliseconds "off" time/100 pulses) for a total energy fluence of 0.1 J/cm.. Gene expression was examined using commercially available extra cellular matrix and adhesion molecule RT PCR Arrays (SA Biosciences, Frederick, MD) at 24 hours post-exposure. RESULTS: Different expression profiles were noticed for each of the ratios studied. Overall, there was an average (in an 80 gene array) of 6% expression difference in up or downregulation between the arrays. The greatest increase in collagen I and decrease in collagenase (MMP-1) was observed with 75/25% ratio of 590/870 nm. The addition of increasing proportions of IR wavelengths causes alteration in gene expression profile. The ratios of the wavelengths caused variation in magnitude of expression. CONCLUSIONS: Cell metabolism and gene expression can be altered by simultaneous exposure to multiple wavelengths of low energy light. Varying the ratios of specific wavelength intensity in both visible and near infrared light therapy can strongly influence resulting fibroblast gene expression patterns.

Metachronous hepatic resection for liver only pancreatic metastases.

BACKGROUND: The value of liver resection (LR) for metachronous pancreatic ductal adenocarcinoma (PDAC) metastases remains controversial. However, in light of increasing safety of liver resections, surgery might be a valuable option for metastasized PDAC in selected patients. METHODS: We performed a retrospective, multicenter study including patients undergoing hepatectomy for metachronous PDAC liver metastases between 2004 and 2015 to analyze postoperative outcome and overall survival. All patients were operated with curative intent. Patients with oligometastatic metachronous liver metastasis with definitive chemotherapy (n = 8) served as controls. RESULTS: Overall 25 patients in seven centers were included in this study. The median age at the time of LR was 63.8 years (56.9-69.9) and the median number of metastases in the liver was 1 (IQR 1-2). There were eight non-anatomical resections (32%), 15 anatomical minor (60%) and 2 major LR (8%). Postoperative complications occurred in eleven patients (eight Clavien-Dindo grade I complications (32%) and three grade IIIa complications (12%), respectively). The 30-day mortality was 0%. The median length of stay was 8.6 days (IQR 5-11). Median overall survival following LR was 36.8 months compared to 9.2 months in patients with metachronous liver metastasis with chemotherapy (p = 0007). DISCUSSION: Liver resection for metachronous PDAC metastasis is safe and feasible in selected patients. To address general applicability and to find factors for patient selection, larger trials are urgently warranted.

The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.

Around-the-World Relativistic Sagnac Experiment.

In 1971 Hafele and Keating carried portable atomic clocks east and then west around the world and verified the Sagnac effect, a special relativity effect attributable to the earth's rotation. In the study reported here observations of the effect were made by using electromagnetic signals instead of portable clocks to make clock comparisons. Global Positioning System satellites transmit signals that can be viewed simultaneously from remote stations on the earth; thus an around-the-world Sagnac experiment can be performed with electromagnetic signals. The effect is larger than that occurring when portable clocks are used. The average error over a 3-month experiment was only 5 nanoseconds.

Molecular basis of mammalian sexual determination: activation of Müllerian inhibiting substance gene expression by SRY.

The pathway of male sexual development in mammals is initiated by SRY, a gene on the short arm of the Y chromosome. Its expression in the differentiating gonadal ridge directs testicular morphogenesis, characterized by elaboration of Müllerian inhibiting substance (MIS) and testosterone. SRY and MIS each belong to conserved gene families that function in the control of growth and differentiation. Structural and biochemical studies of the DNA binding domain of SRY (the HMG box) revealed a protein-DNA interaction consisting of partial side chain intercalation into a widened minor groove. Functional studies of SRY in a cell line from embryonic gonadal ridge demonstrated activation of a gene-regulatory pathway leading to expression of MIS. SRY molecules containing mutations associated with human sex reversal have altered structural interactions with DNA and failed to induce transcription of MIS.

High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.

BACKGROUND: Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS: The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS: The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

The Time Deviation in Packet-Based Synchronization.

The telecommunications industry has used the time deviation (TDEV) very effectively for specifying network equipment clock performance as well as the performance of timing signals generated by Central Office equipment such as primary reference clocks and building integrated timing supplies (BITS) and synchronization supply units (SSUs). We discuss here the development of TDEV, and the variations of TDEV motivated by the advent of packet-switching and the steady transformation of the telecom network from circuit-switched-based to packet-switched-based. We illustrate these with simulation of the performance of the precise time protocol (PTP) across a packet-switched network. We then apply published methods to automatically determine noise types, and use these to predict time dispersion from a master clock for a slave clock using these PTP packets to stay synchronized. The result shows how TDEV and the other deviations provide an extensive array of tools for telecom networks, as well as for general time and frequency applications.

Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition.

Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.

Electrostatics and hydration at the homeodomain-DNA interface: chemical probes of an interfacial water cavity.

Electrostatics and hydration of a homeodomain-DNA complex are dissected by chemical modification. Selective neutralization of phosphate charges by methylphosphonate substitution demonstrates the differential importance of short- and long-range electrostatic interactions. Whereas the footprint of direct contacts is in accord with crystal structures, interference is also observed at non-contacted sites. Such sites adjoin a novel interfacial water cavity in the major groove. Non-contacted phosphodiester groups in the cavity are proposed to contribute to long-range ordering of an extended protein-water-DNA interface. Use of isolated S(p) and R(p) methylphosphonate diastereomers demonstrates that interference at this extended interface is stereoselective and charge-independent. Attenuation of protein binding presumably reflects groove-specific reorganization of bound water. Surprisingly, such attenuation can exceed that due to neutralization of a direct phosphate-side-chain salt bridge. These results support the hypothesis that hydration of an interfacial cavity functions as a non-covalent extension of the DNA surface. Stereo-specific interrogation of bound water by chemical synthesis provides a general method to assess the coupling between solvation and DNA recognition.

The dimerization domain of HNF-1alpha: structure and plasticity of an intertwined four-helix bundle with application to diabetes mellitus.

Maturity-onset diabetes mellitus of the young (MODY) is a human genetic syndrome most commonly due to mutations in hepatocyte nuclear factor-1alpha (HNF-1alpha). Here, we describe the crystal structure of the HNF-1alpha dimerization domain at 1.7 A resolution and assess its structural plasticity. The crystal's low solvent content (23%, v/v) leads to tight packing of peptides in the lattice. Two independent dimers, similar in structure, are formed in the unit cell by a 2-fold crystallographic symmetry axis. The dimers define a novel intertwined four-helix bundle (4HB). Each protomer contains two alpha-helices separated by a sharp non-canonical turn. Dimer-related alpha-helices form anti-parallel coiled-coils, including an N-terminal "mini-zipper" complementary in structure, symmetry and surface characteristics to transcriptional coactivator dimerization cofactor of HNF-1 (DCoH). A confluence of ten leucine side-chains (five per protomer) forms a hydrophobic core. Isotope-assisted NMR studies demonstrate that a similar intertwined dimer exists in solution. Comparison of structures obtained in multiple independent crystal forms indicates that the mini-zipper is a stable structural element, whereas the C-terminal alpha-helix can adopt a broad range of orientations. Segmental alignment of the mini-zipper (mean pairwise root-mean-square difference (rmsd) in C(alpha) coordinates of 0.29 A) is associated with a 2.1 A mean C(alpha) rmsd displacement of the C-terminal coiled-coil. The greatest C-terminal structural variation (4.1 A C(alpha) rmsd displacement) is observed in the DCoH-bound peptide. Diabetes-associated mutations perturb distinct structural features of the HNF-1alpha domain. One mutation (L12H) destabilizes the domain but preserves structural specificity. Adjoining H12 side-chains in a native-like dimer are predicted to alter the functional surface of the mini-zipper involved in DCoH recognition. The other mutation (G20R), by contrast, leads to a dimeric molten globule, as indicated by its 1H-NMR features and fluorescent binding of 1-anilino-8-naphthalene sulfonate. We propose that a glycine-specific turn configuration enables specific interactions between the mini-zipper and the C-terminal coiled-coil.

Comparison of the dynamics of an engineered insulin monomer and dimer by acid-quenched amide proton exchange. Non-local stabilization of interchain hydrogen bonds by dimerization.

Dynamic differences between an engineered insulin monomer and dimer are investigated under physiological conditions by an adaptation of the method of acid-quenched amide proton exchange. Although exchange lifetimes of amide protons involved in intrachain hydrogen bonds are similar in the two analogs, dimerization specifically stabilizes two interchain hydrogen bonds (LeuB6-NH...O = C-CysA6 and CysA11-NH...O = C-GluB4) that are distant from the dimer interface. Such non-local stabilization demonstrates that fluctuations in the tertiary structure of the monomer are damped by dimerization. As the B6-A6 and A11-B4 hydrogen bonds are specific to the crystallographic T-state, their stabilization also indicates that the R-state (an allosteric feature of hexamer assembly) is not significantly populated in an isolated dimer. Our results are discussed in reference to recent hypotheses that crystal structures of insulin depict inactive conformers and that detachment of interchain contacts accompany receptor binding.

Aromatic-aromatic interactions in the zinc finger motif. Analysis of the two-dimensional nuclear magnetic resonance structure of a mutant domain.

The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL apical domain with implications for substrate interactions.

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (Tm>100 degrees C as evaluated by circular dichroism) is at least 35 degrees C greater than that of the E. coli apical domain (Tm=65 degrees C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 A using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 A based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the "Anfinsen cage" in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using 15N-1H correlation spectra of 15N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50 degrees C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal alpha-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.

Structure and dynamics of a protein assembly. 1H-NMR studies of the 36 kDa R6 insulin hexamer.

The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers.

Mapping the functional surface of insulin by design: structure and function of a novel A-chain analogue.

Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.

Native and non-native structure in a protein-folding intermediate: spectroscopic studies of partially reduced IGF-I and an engineered alanine model.

The structure of a metastable folding intermediate of human insulin-like growth factor I (IGF-I) and an engineered model are investigated by circular dichroism and two-dimensional 1H NMR spectroscopy. The intermediate, which contains two of three native disulfide bonds, was trapped by acid quenching and isolated by reverse-phase HPLC. The reduced cysteine residues were mapped to residues 47 and 52 (corresponding to A6-A11 in insulin). In the native state this disulfide bridge anchors an adjoining amphipathic alpha-helix (helix 2; residues 42 to 49) against the hydrophobic core. Comparison of CD and 1H-NMR spectra demonstrates that the acid-quenched intermediate is partially folded and contains elements of native secondary and tertiary structure. Spectra are similar to those of an equilibrium model in which the reduced cysteine residues are replaced by alanine. Complete 1H-NMR sequential assignment of the alanine model has been obtained and demonstrates that removal of the disulfide bond is associated with local unfolding of the adjoining alpha-helix. Native secondary structure (including helices 1 and 3) is otherwise retained and defines a folded subdomain. Long-range nuclear Overhauser effects (NOE) within this subdomain are similar to those of native IGF-I; no non-native NOE is observed. Our results support the hypothesis that folding of the insulin motif is directed by a subset of native structural elements and that these elements form at an early step in the pathway. Formation of helix 2, despite its prominence in the native state, is likely to represent a late step. Hydrophobic collapse of this segment appears to precede helix formation.

Mini-proinsulin and mini-IGF-I: homologous protein sequences encoding non-homologous structures.

Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T3R3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corresponding two-chain analogue. The two proteins each contain substitutions in the B-chain (HisB10-->Asp and ProB28-->Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between LysB29 and GlyA1. The structures are essentially identical, resembling in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 alpha-helix (variable among crystal structures) is less well-ordered in mini-proinsulin than in the two-chain variant. The single-chain analogue is not completely inactive: its affinity for the insulin receptor is 1500-fold lower than that of the two-chain analogue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29-A1 peptide bond, optimizes affinity but is not integral to the mechanism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms deviation 4.5 A) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 A). Three-dimensional profile scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures.