Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D₁-like receptors (D₁R and D₅R) stimulate adenylyl cyclase (AC) activity, whereas the D₂-like receptors (D₂, D₃ and D₄) inhibit AC activity. D₁R, but not the D₅R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D₁R and D₅R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D₁ or D₅ receptor (HEK-hD₁R or HEK-hD₅R) and human renal proximal tubule (hRPT) cells that endogenously express D₁R and D₅R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD₁R (84.5 ± 2.3% of total), HEK-hD₅R (68.9 ± 3.1% of total), and hRPT cells (66.6 ± 2.2% of total) (P < 0.05, n = 4/group). In HEK-hD₁R cells, the D₁-like receptor agonist fenoldopam (1μM/15 min) increased AC5/6 protein (+ 17.2 ± 3.9% of control) in LRs but decreased it in non-LRs (− 47.3 ± 5.3% of control) (P < 0.05, vs. control, n = 4/group). By contrast, in HEK-hD₅R cells, fenoldopam increased AC5/6 protein in non-LRs (+ 67.1±5.3% of control, P < 0.006, vs. control, n = 4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D₁R (− 94.5 ± 2.0% of control) and HEK-D₅R cells (− 87.1 ± 4.6% of control) but increased it in hRPT cells (6.8 ± 0.5-fold). AC6 activity was stimulated to a greater extent by D₁R than D₅R, in agreement with the greater colocalization of AC5/6 with D₁R than D₅R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D₁R- and D₅R-mediated AC signaling.     

Thiourea mediated regulation in the expression profile of aquaporins and its impact on water homeostasis under salinity stress in Brassica juncea roots

Bioregulatory molecules such as thiourea (TU) play an important role in imparting stress tolerance to crops. However, the molecular mechanism involved in the TU-mediated tolerance has not been elucidated. Towards this endeavour, the expression profile of various PIPs (plasma membrane intrinsic proteins) was studied under salt stress (NaCl; 700 mM) with/without thiourea (TU; 6.5 mM) at different time periods in roots of Brassica juncea. Various aquaporin isoforms demonstrated an upregulation upon salinity stress imposition, whereas they were downregulated upon TU supplementation. TU treatment also led to a decrease in the accumulation of reactive oxygen species and delimited the need for an enhanced accumulation of osmolytes. The vacuolar pH was also maintained in NaCl + TU treatment as demonstrated by in vivo ³¹P NMR of roots. In conclusion, TU supplementation to salt stressed seedlings was found to maintain the water homeostasis of roots through coordinated regulation of different PIP isoforms.     

Measurement of waist circumference for retrospective studies – Prospective validation of use of CT images to assess abdominal circumference

IntroductionTo validate the use of supine position and CT images for assessing abdominal circumference (AC).MethodA prospective study in consecutive patients undergoing scheduled abdominal CT at our center between 17 and 25 September 2012.AC was measured four times:

• 1.Standing.
• 2.While lying on the CT table.
• 3.On CT images with a skin contour line, using OsiriX software.
• 4.On CT images with an ellipse perimeter formula, using RAIM Alma 2010 software.

Measurements 1 and 2 were sequentially done by the same trained nurse before abdominal CT just above the iliac crest, while measurements 3 and 4 were done on the last abdominal CT slice not showing the iliac bone. Student's t tests and Q-Q and Bland–Altman plots were used for statistical analysis.ResultsA total of 102 patients were recruited. Mean age, 60 (35–78) years. Mean BMI, 25 (18–39) kg/m². Mean AC, 93.2 (73–135) cm.No significant differences were found between the four ACs measured (Student's t test, P = 0.83).Q-Q and Bland–Altman plots showed good overlapping for the low and central values (73–110 cm) with a greater scatter for extremely high values.For the ellipse estimation, R² was 0.987 with a mean error of 0.4 cm and a stretch dispersion between 1.1 and −0.3 cm.ConclusionSupine (either measured or estimated on CT images by free hand elliptical ROI or ellipse formula) and standing measurements appear to be equivalent for abdominal circumferences <110 cm.     

Tetracycline-regulatable expression vectors tightly regulate in vitro gene expression of secreted proteins

The regulation of gene expression by the tetracycline system has attracted a high level of interest in the recent past. However, expression of secreted proteins has not been evaluated precisely. In this study, we constructed two versions of a one-plasmid system containing the elements necessary for the regulation of gene expression. The regulatable elements and the selectable marker (Neo^r) were set up in two different configurations, pTRIN31 and pTRIN76. With these two regulatable versions, the levels of protein expression after transfection into the NIH/3T3 cell line were measured by insertion of three different genes encoding the secreted proteins (hGH, ApoE3, hGM-CSF). The maximum levels of gene expression obtained with the pTRIN76-derived plasmids were 100 ng/24 h/10⁶ cells for hGH, 427 ng/24 h/10⁶ cells for ApoE3 and 108 ng/24 h/10⁶ cells for hGM-CSF. For the pTRIN31-derived plasmids the maximum levels were 2.7 ng/24 h/10⁶ cells for hGH and 47 ng/24 h/10⁶ for ApoE3. Both plasmids give rise to an expression of the transfected gene that can be tightly regulated by three different molecules: tetracycline, minocycline and doxycycline. The levels of the secreted proteins are below the detectable level when the reporter genes are repressed. This repression is reversible within 48 h after the regulator has been removed from the medium.     

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.     

6-Triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3-triazol-4-yl-/5-yl moieties were synthesized by employing click chemistry. The new sulfocoumarins incorporated cycloalkyl, tert-butyl and substituted aryl moieties at the triazole ring, and were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The triazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms hCA I and II (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 7.2 to 10.5 nM against hCA IX, and between 5.5 and 17.7 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Coumarins incorporating hydroxy- and chloro-moieties selectively inhibit the transmembrane,tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II

A series of coumarins incorporating hydroxy-, chloro- and/or chloromethyl-moieties in positions 3-, 4-, 6- and 7- of the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and XII. The nature and position of the groups substituting the coumarin ring greatly influenced CA inhibitory properties. 6-Hydroxycoumarin showed K_Is >100 μM against CA I and II, of 0.198 μM against CA IX and of 0.683 μM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic isoforms. These compounds are also excellent leads for designing isoform-selective enzyme inhibitors.     

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K⁺) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K⁺ supplementation (1.31 ± 0.04 mmol/L and 2.06 ± 0.04 mmol/L K⁺; mean ± SEM), one was maintained with crystal salt and no K⁺ supplementation (0.33 ± 0.05 mmol/L K⁺), the fourth system was maintained using a standard marine mix salt (2.96 ± 0.04 mmol/L K⁺), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na⁺/K⁺-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na⁺/K⁺/2Cl^? cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K⁺ supplementation resulted in 100% mortality within 24 h. Mortality and dry mass were significantly influenced by K⁺ concentration (P ≤ 0.05). No differences were observed among treatment groups for NKA activity. At 1 dph NKA mRNA expression was higher in the 0.3 mmol [K⁺] group than in other treatment groups and at 7 dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.     

Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1 = isoform 2 < isoform 4 < isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM against hCA IX and of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.     

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): Purification,primary structure,and inhibitory properties

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K_a = 1.1 × 10⁹ M^?1 and 2.5 × 10⁹ M^?1, respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K_a = 4.5 × 10⁸ M^?1 and 1.2 × 10¹⁰ M^?1). Weak interaction with human plasmin (K_a = 1.2 × 10⁷ M^?1) was also revealed.     

Design,synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with K_Is ranging between 11 and 390 nM, whereas they were inactive against both CA I (K_Is >50 μM) and II (K_Is ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.     

Impact of genotype,plant growth regulators and activated charcoal on embryogenesis induction in microspore culture of pepper (Capsicum annuum L.)

The production of double haploids through androgenesis is used by breeders to produce homozygous lines in a single generation. Androgenesis can be achieved by isolated microspore culture, which, however, allows the production of embryogenesis with a very low efficiency. In order to improve the overall embryogenesis in pepper, we study the differences of microspore embryogenesis in different genotypes of pepper, and also document the effect of growth regulators in pretreatment media, and activated charcoal (AC) on embryogenesis induction. Fifty different pepper genotypes were evaluated, and the swollen rate of microspores from different genotypes varied from 3.11% to 29.56% with the mean value of 13.13%. Microspores from genotype ‘36’ had the highest swollen rate, and the lowest swollen rate of microspores was observed in genotype ‘26’. It was concluded from the statistical results of L₉ (3³) orthogonal test that changes in the level of BA influenced the swollen rate of microspores more significantly, and the combination of 0 mg∙l^− 1 6-benzyladenine (BA), 0.2 mg∙l^− 1 α-naphthaleneacetic acid (NAA) and 0.5 mg∙l^− 1 kinetinin (Kin) was best. AC at a concentration of 0.05% could act as a promoter of embryogenesis in the microspore culture of different pepper genotypes, while the more significant effect was observed with the low responsive genotypes.     

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.     

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues,heart failure and atherosclerosis

The sarco(endo)plasmic reticulum Ca²⁺ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis).Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~ 99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~ 91% of total SERCA and SERCA2b for ~ 5%. Among SERCA3, SERCA3b was the most expressed (~ 3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b.In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively.In conclusion: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~ 30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation–contraction coupling in both CMs and contractile VSMCs.     

The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium

STIM1 acts as an endoplasmic reticulum Ca^2 + sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca^2 + binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca^2 + concentrations in store-operated Ca^2 + entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca^2 + in a concentration-dependent manner. Expression of the Ca^2 +-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca^2 +. Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn^2 + entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca^2 +, which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca^2 + mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca^2 + concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, VII,IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here.     

De novo design of transmembrane helix–helix interactions and measurement of stability in a biological membrane

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu “scaffold” was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix–helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔG^app) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (< 1.4 kcal mol^− 1) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).