1 Supporting information for Protein Conformational Flexibility Enables the Formation of Dense Liquid Clusters: Tests Using Solution Shear Michael C. Byington a , Mohammad S. Safari a , Jacinta C. Conrad a, *, and Peter G. Vekilov a,b, * a Department of Chemical and Biomolecular Engineering and b Department of Chemistry, University of Houston, 4726 Calhoun Road, Houston, TX, 77204-4004, USA. Materials and Methods Solution preparation. Lysozyme (Thermo Scientific #89833) used for these experiments was dissolved in DI water (18.2 MΩ by Millipore MilliQ Gradient) containing 1.5mM sodium azide (Sigma 438456), and, if indicated, 20mM HEPES (Sigma Aldrich #H3375) buffer. The solution was dialyzed against 1 L of the respective solvent using dialysis cassettes with 2000 g mol -1 cutoff (Life Technologies Corp #66203) for 38-48 hours in a refrigerator (4 o C) on a stir plate at ~50 rpm with a 2” magnetic stir bar. The beaker (1-2 L) used for dialysis was covered with parafilm (Bemis Flexible Packaging #PM996) to prevent evaporation. The solution was removed from the dialysis cassettes with 10 mL syringes and 18 gauge needles (Becton Dickinson #309604 and 305196 respectively) and filtered through 30 mm 220 nm pore size syringe filters (LightLabs TC-9747). The concentration was determined by diluting an aliquot of the prepared solution 1000-fold and measuring the absorption at 280 nm using a Beckman DU-800 spectrophotometer. The blank used HEPES or plain DI water that match the solvent. Shear experiments were performed immediately after solution preparation using a shear cell constructed as in Figure 1 with 20 mL glass syringe (Grainger 19G353) and 5/8” Teflon rod (Grainger 30GC44). To ensure rod remained centered at the bottom of the syringe, the end of the rod was machined to fit the inner tip profile of the syringe. Teflon rod was rotated by a DC motor (Amico Model RC385SAP-2173-57) and DC power source (Dr. Meter HY1803D). The shear rate was set by voltage applied to the DC motor. Shear rate and voltage were calibrated by counting the rotations in a 60 sec interval at various voltages (1.5 – 11 V, 0.5 volt increments) then computing the shear rate in the bulk fluid assuming a linear velocity profile and no slip boundary conditions. Shearing started immediately after solution preparation. In experiments using mercaptoethanol or urea, the respective reagent was added immediately before the start of shearing. Every hour, a 300 μL solution aliquot was removed from the shear cell with a 1 mL syringe and an 18 gauge needle (Becton Dickinson #309628 and 305196 respectively). This solution sample was filtered through a 13 mm 220 nm cutoff syringe filter (LightLabs TC-9746) directly into a DLS cuvette (Fisherbrand 14-961-25), capped with parafilm, and loaded into the sample holder of the light scattering device. Light scattering date were collected on an instrument byALV-GmbH, Langen, Germany, equipped with He-Ne laser operating at 632.8 nm, and an ALV-5000 EPP Multiple tau Digital Correlator). At least 10 correlation functions of 60 seconds were collected. To extract the average cluster radius R 2 and fraction of the solution volume occupied by the cluster population  2 from DLS data, all correlation functions were fitted to: 1 [ 2 () − 1] 2 = 1 exp (−   1 )+ 2 exp (−   2 )+