Molecular Cloning and Protein Expression
The open reading frame of rat Ric-8A and truncation variants (encompassing residues 1–492, 12–492, 1–453, 1–426, 1–402, 1–374, 12–492 and 38–492) were amplified by PCR and subcloned into the pET-28a vector for expression as N-terminally hexa-histidine tagged proteins. Proteins were expressed in Escherichia coli BL21 (DE3)-RIPL cells in LB media containing ampicillin (120 mg/L) and induced with 300 µM isopropyl β-d-thiogalactopyranoside (IPTG) at 20°C. After overnight growth at 20°C, cells were lysed by sonication at 20°C in lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 2 mM DTT, and 2 mM PMSF. The cell lysate was clarified by centrifugation and loaded onto a column containing 5 ml of nickel NTA-agarose (GE Healthcare). After extensive washing with lysis buffer, proteins were eluted from the resin with buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 2 mM PMSF) containing 250 mM imidazole and dialyzed in a low ionic strength buffer (50 mM Tris, pH 8.0, 2 mM DTT, and 2 mM PMSF). The dialysate was loaded onto a UNO-Q matrix (Bio-Rad) and eluted with a 0–500 mM NaCl gradient on an AKTA FPLC system (GE Healthcare). Pure Ric-8AΔC492 eluted from the matrix at 165–175 mM NaCl.
Rat Gαi1 was expressed as a tobacco etch virus protease (TEV)-cleavable, N-terminal glutathione-S-transferase (GST) fusion protein as described 
. W258A-Gαi1, in which the tryptophan residue at position 258 is substituted by alanine, was generated by use of the QuikChange (Stratagene) kit according to the manufacturer's protocol, using the pDEST-15 vector harboring wild type GST-Gαi1 as a template. To generate NΔ25-Gαi1, from which the N-terminal 25 residues of the native protein are deleted, attB-modified primers corresponding to amino acids 25 to 35 and 343 to 353 of Gαi1 were used for PCR amplification and cloning of the fragment into the pDEST15 vector. W258-Gαi1 and NΔ25-Gαi1 were expressed and purified as described 
The plasmid pBN905, which expresses rat Gαi1ΔC9, lacking the C-terminal nine residues of the native protein, fused in-frame to intein-CBD cDNA in the pTXB3 expression vector (New England Biolabs), was a kind gift from Dr. T.J. Baranski, Washington University, St. Louis, MO. Gαi1ΔC9 was expressed and purified as described 
. With the exception of experiments summarized in , and , all other experiments were performed with Ric-8AΔC492 and W258A-Gαi1, which we henceforth refer to as Ric-8A and Gαi1, respectively.
Nucleotide-free Gαi1 proteins were prepared by the method of Ferguson and Higashijima 
, using exchange and dialysis buffers composed of 50 mM Tris.HCl, pH 8.1, 2 mM Tris (2-carboxyethyl)phosphene (TCEP) with 20% glycerol (v/v) and 150 mM NaCl. GTPγS-bound Gαi1 was prepared as described 
Preparation of 15N-labeled proteins
N]Gαi1 was prepared as described with minor modifications 
. Briefly, transformed E. coli
cells were grown in minimal media supplemented with [15
Cl (Cambridge Isotopes, 99.8% purity) and [15
N]Bioexpress Cell Growth media (10 ml of 10× concentrate/liter of media) (Cambridge Isotope Labs), induced with 500 µM IPTG at 20°C and allowed to express Gαi1 overnight at the same temperature. The purification protocol was identical to that used for native proteins and the yields were approximately one third lower.
Preparation of Ric-8A:Gαi1 complexes
Nucleotide-free Ric-8A:Gαi1 and Ric-8A:[15N]Gαi1 complexes were generated by incubating equimolar concentrations of Ric-8A (500 µl of 150 µM protein) with Gαi1•GDP or [15N]Gαi1 (500 µl of 150 µM protein) overnight in sample buffer (20 mM Tris•HCl, pH 8.0, 150 mM NaCl, 2 mM DTT, and 5 mM EDTA) containing 50 µl of immobilized alkaline phosphatase (Sigma) to hydrolyze released nucleotide, and gently rocked at 4°C. The immobilized alkaline phosphatase was removed by centrifugation, and complex was gel-filtered over tandem Superdex 200/75 gel filtration columns pre-equilibrated in 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM DTT and eluted at a flow rate of 0.4 ml/min using an AKTA FPLC (GE Healthcare).
Trypsin Protection Assays
To samples containing Ric-8A, Gαi1•GDP, Gαi1[ ] or Ric-8A:Gαi1 (50 µM in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 2 mM DTT), L-1-p
-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) was added at a molar ratio of 1000
1. Samples were incubated at 4°C for 5, 10, 15 and 25 minutes. For each time point, a 10 µl aliquot was withdrawn, diluted in SDS-PAGE loading buffer, boiled, separated by SDS-PAGE and visualized by Coomassie staining. Proteolytic products were eluted from the gel slices and subjected to MALDI-TOF mass spectrometry on a Voyager DE3 (Applied Biosystems) or by electrospray mass spectometry using on a Agilent 6520 QTOF. Limited trypsinolysis for mass spectrometric identification of large Ric-8A fragments () was conducted in the presence of a 1
2 molar ratio of bovine pancreatic aprotinin to TPCK-trypsin. Proteolytic products were eluted from the gel slices and subjected to electrospray mass spectrometry and N-terminal sequencing at the Protein Chemistry Core Facility of the University of Texas Southwestern Medical Center.
Yeast two hybrid experiments
A rat brain yeast two-hybrid prey library 
was screened with a pVJL11 
bait construct encoding the amino-terminal 297 amino acids of Ric-8A (1–297) in the L40 yeast strain 
with a prey clone consisting of the carboxyl-terminal 81 amino acids of Gαi1 (Gαi1 C-term). Full-length Gαi1 and Gαq, and the Gαi1 C-term preys, in pGADGH (Clonetech) were then tested pair-wise using a β-galactosidase filter assay for interactions with the full-length Ric-8A bait (amino acids, 1–530 FL), and truncated Ric-8A bait constructs that coded for different regions of the Ric-8A protein (amino acids: 115–530, 1–297, 1–247, 67–297), or the empty prey vector.
[35S]GTPγS Binding Assays
Binding of [35
S]GTPγS to wild-type Gαi1 or W258A-Gαi1, in the presence or absence of Ric-8A was performed using a filter binding method 
in a 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM DTT and 0.05% polyoxyethylene lauryl ether (C12E10).
Pull-down assays for Gαi1 binding to Ric-8A fragments
Equimolar amounts of Ric-8A and Gαi1 (10 µM protein in 50 mM TRIS. HCl, pH 8.0, 150 mM NaCl, 2 mM DTT and 0.05% C12E10) were incubated overnight at 4°C. 10 µl of a 50% slurry of Ni+2 IMAC (BioRad) resin was then added to the mixture, incubated for one hour, washed thrice with 500 µl of wash buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM DTT and 2 mM PMSF) and the proteins retained on the beads were visualized by Coomassie stained SDS-PAGE.
An amidated peptide, Gαi1C18, corresponding to the C-terminal 18 residues of rat Gαi1 (DAVTDVIIKNNLKDCGLF) was synthesized using standard FMOC chemistry by the Protein Chemistry Core laboratory at UT Southwestern Medical Center at Dallas and purified to near homogeneity by HPLC (Agilent Technologies) on a pre-packed C18 matrix (Waters). Mass of the peptide was confirmed by MALDI-TOF mass spectrometry (Voyager DE, Applied Biosystems).
Peptide competition assays
Exchange of GTPγS for GDP bound to Gαi1 or Gαi1CΔ9 was followed by monitoring the change in the tryptophan fluorescence of Gαi1, as described 
. Gαi1•GDP (1 µM) in 20 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM MgCl2
, 1 mM DTT, and 0.05% C12E10 in a reaction volume of 400 µl was allowed to equilibrate for 10–15 min at 20°C in a quartz fluorescence cuvette. GTPγS (final concentration, 10 µM) was added to the reaction mixture in the absence or presence of 1 µM Ric-8A, and the increase in fluorescence at 340 nm was monitored upon excitation at 290 nm 
. Exchange kinetics were also measured in the presence of 1 µM Ric-8A and (5–50 µM) Gαi1C18. Protein and peptide mixtures were preincubated for one hour before addition of GTPγS. Fluorescence measurements were conducted using an LS55 spectrofluorometer (PerkinElmer Life Sciences) attached to a circulating water bath to maintain a steady sample temperature of 20°C. Excitation and emission slit widths were set at 2.5 nm. All exciting light was eliminated by use of a 290 nm cut-off filter positioned in front of the emission photomultiplier.
Circular Dichroism spectroscopy
Gαi1•GDP, Ric-8A, Gαi1[ ] or the nucleotide free binary complex of the two proteins at 4 µM each in 25 mM HEPES, pH 7.2, 150 mM NaCl and 2 mM DTT and, in the case of Gαi1[ ], 20% v/v glycerol, were dispensed into a 300-µl quartz cuvette with a 1 mm path length. CD spectra in the range of 195–245 nm were measured at a scan rate of 1 nm/min using a PiStar-180 CD spectrometer (Applied Photophysics). The scans were repeated thrice; the data were averaged and the CD spectra of the buffer was subtracted. The optical path and the cuvette chamber were continually flushed with a nitrogen flow throughout the course of the experiment. Secondary structure analysis was performed using K2D2 
Protein samples for NMR spectroscopy ([15
N]Gαi1:Ric-8A or [15
N]Gαi1•GTPγS) were dialyzed against 20 mM sodium phosphate, pH 6.8, 75 mM NaCl and 2 mM DTT in 10% 2
O, and concentrated to 250 µM. The [15
N]Gαi1•GTPγS sample was prepared from the [15
N]Gαi1:Ric-8A complex by addition of five molar excess of GTPγS and incubation for 10 minutes at 25°C. Free Ric-8A, and non-dissociated [15
N]Gαi1:Ric-8A complex were then removed with Ni+2
IMAC resin (BioRad). 1
N TROSY-HSQC spectra 
were acquired at 25°C on a 600 MHz Varian NMR System equipped with a salt-tolerant cold probe and processed with Felix 2004 (Felix NMR, Inc.).
Hydrogen-Deuterium Exchange Mass Spectrometry
Hydrogen-deuterium exchange of the Gαi1•GDP or Ric-8A:Gαi1[ ] was analyzed by automated reverse-phase HPLC coupled to electrospray ionization TOF mass spectrometry. The HPLC consisted of an Agilent 1100 HPLC with a G1377a autosampler, and the ESI-TOF was a Bruker microTOF. Following initiation of the reaction by ten-fold dilution of protein stock (1 mg/ml Gαi1•GDP or Ric-8A:Gαi1[ ], in 20 mM sodium phosphate, pH 6.8, 100 mM NaCl and 1 mM DTT) into D2O, the reaction mixture was pipetted into a sealed autosampler vial and the autosampler was used to draw aliquots at regular time intervals. Quenching of the exchange reaction was achieved by rapid binding of the protein onto a C4 reverse phase cartridge from Michrom Bioresources (8×1 mm) and subsequent washing and elution. The column and autosampler were pre-equilibrated with 20% (v/v) acetonitrile, 80% H2O and 0.1% formic acid (w/v), pH 2.2, prior to sample loading. Immediately following sample (0.5 µl) injection, the solvent composition was changed to 100% acetonitrile, 0.1% formic acid. By using a rapid step gradient and very high flow rates of 600 µl/min, the sample was minimally delayed in the flow path to the mass spectrometer, eluting at approximately 0.4 minutes. The column system was equilibrated at 4°C to minimize back-exchange. Data processing was performed with the Bruker Data Analysis software package, version 4.0. The Maximum Entropy devolution routine was used to perform charge-deconvolution for the spectral range of 700 m/z to 1400 m/z, which encompassed the majority of the observed distribution of protein signal. The deconvoluted spectra were exported to ORGIN software and the centroid masses for Gαi1 were calculated and plotted as a function of time.
Differential Scanning Calorimetry (DSC)
For DSC analysis, Gαi1•GDP, Ric-8A and Ric-8A:Gαi1[ ] were dialyzed against degassed DSC sample buffer: 25 mM PIPES pH 7.2, 150 mM NaCl and 1 mM TCEP, and additionally for the Gαi1•GDP sample, 20 µM GDP. DSC buffer for Gαi1[ ] contained 20% glycerol (v/v). Immediately before DSC analysis, protein samples were clarified by centrifugation at 14,000 RPM for 10 min in a bench-top Eppendorf microfuge. Protein concentrations after dilution, if required, were determined by least squares fitting of predicted protein extinction coefficients to spectra in the 220–420 nm range measured on a HP diode array instrument. The measured values were 3.6 µM for Gαi1•GDP, 5.9 µM for Ric-8A, 5.1 µM for Ric-8:Gαi1[ ] and 7.9 µM for Gαi1[ ]. DSC measurements were conducted using a Microcal capDSC with autosampler (MicroCal, GE Healthcare). After establishing a thermal history by running water vs. water scans, three buffer against water scans were conducted for each sample using the corresponding dialysate solution to obtain the buffer Cp over the experimental temperature range. Following this, two buffer vs protein scans were performed. Protein samples were rescanned once to check for thermal reversibility.
A typical thermal cycle involved cooling the instrument to 20°C after which a 10 min. thermal equilibration was initiated. Following thermal equilibration, scanning of the sample was performed at a scan rate of 1°C/min over a 20°C–70°C range using passive feedback gain mode and a filtering period of 5 seconds. Once the experimental high temperature limit was reached (70°C), the instrument was cooled back to the starting temperature.
Data analysis was performed using Origin 7.0 by first subtracting the last buffer scan from the protein thermal scan. After normalizing the data to the protein concentration, a progressive baseline estimation was performed by calculating the fractional contribution of the native and denatured state to the sample Cp at each point beneath the excess heat capacity function, thus producing a smoothly varying function of temperature 
. Data presented in were corrected by subtraction of the temperature-dependent change in Cp of the buffer. A weighted average thermal profile for Gαi1[ ] and Ric-8A was computed using the expression CpAv
(T), where wRic-8A
and wGαi1[ ]
are weighting factors for Ric-8A and Gαi1[ ] contributions to the heat capacity at temperature T, and CpRic-8A
and CpGαi1[ ]
are the heat capacities measured for free Ric-8A and Gαi1[ ] at that temperature.