NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys3—Cys31, Cys5—Cys20, and Cys10—Cys30) in the oxidized form (HD5ox). To probe the solution structure and oligomerization properties of HD5ox, and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays. The NMR solution structure of HD5ox, solved at pH 4 in 90:10 H2O/D2O, is presented (PDB: 2LXZ). Relaxation T1/T2 measurements and the rotational correlation time (Tc) estimated from a [15N,1H]-TRACT experiment demonstrate that HD5ox is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19-21 (Val19-Cys20-Glu21) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7-14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5ox is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14,363 Da were determined for HD5ox at pH 7, supporting a tetrameric form ([HD5ox] ≥ 30 μM). At pH 2, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7,079 Da, corresponding to a HD5ox dimer, were obtained. Millimolar concentrations of NaCl, CaCl2, and MgCl2 have negligible effect on the HD5ox sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environment-sensitive behavior of HD5ox in solution, and provide important insights into HD5ox structure/activity relationships and the requirements for antimicrobial action. Host-defense peptides and proteins are key players in the mammalian innate immune response, and serve to prevent colonization by invading pathogenic microbes.1-5 Human defensins are ribosomally-synthesized, cysteine-rich, host-defense peptides expressed in neutrophils (human neutrophil peptides, HNPs) and various types of epithelial cells (αand β-defensins).6-9 Human defensin 5 (HD5), the focus of this work, is an α-defensin comprised of thirty-two amino acids that exhibits three regiospecific disulfide bonds with the connectivities Cys3—Cys31, Cys5—Cys20, and Cys10—Cys30 in the oxidized form, *Corresponding author: [email protected], Phone: 617-452-2495, Fax: 617-324-0505. SUPPORTING INFORMATION AVAILABLE Tables S1-S9 and Figures S1-S47. This information is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author Manuscript Biochemistry. Author manuscript; available in PMC 2013 December 04. Published in final edited form as: Biochemistry. 2012 December 4; 51(48): 9624–9637. doi:10.1021/bi301255u. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript hereafter HD5ox (Figure 1). Like other α-defensins, the HD5ox disulfide array confers a three-stranded β-sheet structure10 and protease resistance.11,12 HD5 is expressed in the human gastrointestinal,13-16 reproductive,17 and urinary18 tracts. Small intestinal Paneth cells,19 which reside at the base of the crypts of Lieberkühn throughout the small intestine and serve to protect the intestinal epithelium and stem cells from invading microbes, package the HD5 propeptide in subcellular granules.15,20 The 75aa propeptide is converted into the 32-aa mature form by trypsin-catalyzed proteolysis of the N-terminal 43-aa pro region, and HD5 is released into the intestinal lumen in response to microbial invasion.21 Numerous in vitro studies demonstrated that HD5ox exhibits antimicrobial activity against a variety of Gram-negative and -positive human pathogens including Escherichia coli, Salmonella enterica, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus facieum.10,22,23 A HD5 transgenic mouse, which expresses HD5 only in the small intestinal Paneth cells, survived oral Salmonella challenge (1.5 × 109 cfu/mL) at levels that were lethal for the wild-type mouse.24 This observation supports an antibacterial role for HD5 in vivo. Recent HD5 transgenic mouse studies of the commensal microbiota revealed that HD5 expression modulates the composition of the resident microflora.25 Defensin deficiency has been observed in patients with inflammatory diseases of the small bowel.26 A single R13H point mutation in HD5 was observed in a Crohn’s disease patient, and this mutation afforded attenuated cell killing for some bacterial species in vitro.27 Indeed, an E. coli Nissle 1917 strain engineered to biosynthesize and secrete HD5 was recently reported as a possible probiotic therapy for Crohn’s disease and other inflammatory diseases of the bowel.28,29 Antiviral activities of HD5ox are also documented.30-32 For instance, HD5ox blocks infection by various non-enveloped human viruses including adenoviruses31,32 and sexually transmitted papillomaviruses,30 and may provide a natural barrier to certain viral diseases in the female reproductive system. The broad-range antibacterial and -viral activities of HD5ox, in addition to other putative physiological roles, motivate investigations of structure-activity relationships. To date, these studies have addressed the importance of the arginine residues,27 the role of the canonical salt bridge formed by Arg6—Glu14,11 and the disulfide array.12,33 The antibacterial activity of the D-enantiomer, prepared by solid-phase peptide synthesis, was also evaluated and exhibited species-specific activity.34 A recent alanine scan identified Leu29 as a critical determinant for antibacterial activity.35 Taken together, these investigations overwhelmingly support a model whereby the mechanism of HD5ox action differs for Gram-negative (e.g. E. coli) and -positive (e.g. S. aureus) organisms. Whereas a variety of HD5 mutant peptides, including the D-enantiomer and disulfide deletion mutants, retain activity against E. coli, the ability of these peptides to kill S. aureus is severely attenuated.12,33,34 HD5ox disrupts the Gram-negative inner membrane;12 however, the precise details of its mechanism of action against E. coli and other Gram-negative organisms, in addition to how it acts on Grampositive species, are unclear. Extensive mutagenesis studies of the human neutrophil αdefensin HNP136-40 and the murine Paneth cell α-defensin cryptdin-4 (Crp4)41-46 have been presented. In total, these studies delineate that defensin structure/activity relationships must be considered on a case-by-case basis, and highlight the importance of evaluating both electrostatics and hydrophobicity when considering the antimicrobial and -viral activities of human α-defensin peptides.4 We previously reported a HD5ox mutant peptide family where pairs of Cys residues involved in native disulfide linkages were systematically mutated to Ser/Ala residues.12 Many of these mutants retained antibacterial activity against E. coli ATCC 25922 whereas none provided activity against S. aureus ATCC 25923 over the concentration range tested. In addition, removal of one or more disulfide bonds markedly attenuated protease resistance. We therefore hypothesized that the lack of antibacterial activity observed for the mutant Wommack et al. Page 2 Biochemistry. Author manuscript; available in PMC 2013 December 04. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript peptides against S. aureus may result from (i) mutant peptide instability under the assay conditions, (ii) disruption of quaternary structure, an/or (iii) failure to interact with a specific and as-yet unidentified cellular target. Herein we address these possibilities and report extensive biophysical studies designed to probe the solution structure and dynamics of HD5ox and select disulfide mutant peptides (Figure 1). We present the NMR solution structure of native HD5ox in addition to NMR studies of N-HD5[Ser]ox, N-HD5[Ser]ox, and N-HD5red. We also describe the quaternary structure of HD5ox and disulfide mutants by using a combination of NMR dynamics measurements, rotation correlation time measurements, and analytical ultracentrifugation. These investigations demonstrate that the native disulfide array is essential for HD5ox quaternary structure, and that the HD5ox oligomerization state in aqueous solution is condition-dependent. EXPERIMENTAL PROCEDURES Materials and General Methods All solvents, reagents, and chemicals were purchased from commercial suppliers and used as received unless noted otherwise. Deuterated water (D2O), 15N-ammonium chloride, and U-13C-glucose were purchased from Cambridge Isotopes (Cambridge, MA). All aqueous solutions, buffers, and NMR samples were prepared with Milli-Q water (18.2 mΩcm−1) that was passed through a 0.22 μm filter before use. Unlabeled HD5 and mutant peptides were overexpressed as His6-fusion proteins in E. coli BL21(DE3) and were purified as previously described.12 General Instrumentation Analytical and semi-preparative high-performance liquid chromatography (HPLC) were performed on an Agilent 1200 instrument equipped with a thermostated autosampler set at 4 °C and thermostated column compartment generally set at 20 °C, and a multi-wavelength detector set at 220 and 280 nm (500 nm reference wavelength unless noted otherwise). Preparative HPLC was performed using an Agilent PrepStar 218 instrument outfitted with an Agilent ProStar 325 UV-Vis dual-wavelength detector set at 220 and 280 nm. A Clipeus C18 column (5 μm pore, 4.6 × 250 mm, Higgins Analytical, Inc.) set at a flow rate of 1 mL/ min was employed for all analytical HPLC experiments. A ZORBAX C18 column (5 μm pore, 4.6 × 250 mm, Agilent Technologies, Inc.) set at a flow rate of 5 mL/min was employed for all semi-preparative-scale HPLC purification. A Luna 100 Å C18 LC column (10 μm pore, 21.2 × 250 mm, Phenomenex) operated at 10 mL/min was utilized for all preparative-scale HPLC purification. HPLC-grade acetonitrile (MeCN) and HPLC-grade trifluoroacetic acid (TFA) were routinely purchased from EMD. For all HPLC separations, solvent A was 0.1% TFA/H2O and solvent B was 0.1% TFA/MeCN. These solvents were passed through a 0.2-μm filter prior to use. High-resolution mass spectrometry was performed by using an Agilent LC/MS system comprised of an Agilent 1260 series LC system outfitted with an Agilent Poroshell 120 EC-C18 column (2.7 μm pore size) and an Agilent 6230 TOF system housing an Agilent Jetstream ESI source. LC/MS-grade MeCN containing 0.1% formic acid and LC/MS-grade water containing 0.1% formic acid were obtained from J. T. Baker. For all LC/MS analyses, solvent A was 0.1% formic acid/H2O and solvent B was 0.1% formic acid/MeCN. The samples were analyzed by using a gradient of 5-95% B over five min with a flow rate of 0.4 mL/min. The MS profiles were analyzed and deconvoluted by using Agilent Technologies Quantitative Analysis 2009 software version B.03.02. A BioTek Synergy HT plate reader outfitted with a calibrated BioTek Take3 Multi-Volume Plate was employed for optical absorption measurements. Peptide stock solution concentrations were routinely quantified by using the calculated extinction Wommack et al. Page 3 Biochemistry. Author manuscript; available in PMC 2013 December 04. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript coefficients for HD5ox or mutant peptide (Table S1, Supporting Information). Solution and buffer pH values were verified by using either a Mettler Toledo S20 SevenEasy pH meter or a HANNA Instruments HI 9124 pH meter equipped with a microelectrode. Overexpression and Purification of 15N and 13C,15N-Labeled Peptides The plasmids employed for the overexpression of His6-Met-HD5, His6-Met-HD5[Ser], and His6-Met-HD5[Ser] are based on the pET-28b expression vector and are described elsewhere.12 Each expression plasmid was transformed into homemade chemicallycompetent E. coli BL21(DE3) cells and freezer stocks were prepared from single colonies. For large-scale overexpression of 15N-labeled peptides, a 50-mL overnight culture was prepared by inoculating LB media containing 50 μg/mL kanamycin from a freezer-stock of the desired E. coli overexpression strain. The starter culture was grown for 16 h (37 °C, 175 rpm) and the OD600 recorded to confirm that the cultures reached saturation (OD600 ~ 1.5). Aliquots (20 mL) of the overnight culture were centrifuged (3,600 rpm × 10 min, 4 °C) and the supernatant was discarded. The resulting cell pellets were resuspended in 3 mL of sterile-filtered 15N-labeled M9 minimal medium (6.0 g/L disodium phosphate, 3.0 g/L monopotassium phosphate, 0.5 g/L sodium chloride, 1.0 g/L 15N-labeled ammonium chloride) supplemented with 2 mL/L of 1 M MgSO4, 2 mL/L of 5 mM FeCl3, 100 μL/L of 1M CaCl2, 1 mL/L of glycerol, 2.0 g/L of D-glucose, 1 mL/L of 50 mg/mL kanamycin, and 200 μL of a vitamin mix.47 The vitamin mix contained choline chloride (200 mg), folic acid (250 mg), pantothenic acid (250 mg), nicotinamide (250 mg), myo-inositol (50 mg), pyridoxal hydrochloride (250 mg), thiamin hydrochloride (250 mg), riboflavin (25 mg), adenosine (50 mg), and biotin (50 mg) suspended in 7.5 mL of sterile-filtered Milli-Q water. The resuspended bacterial cell pellet was used to inoculate 1 L of the same minimal medium and the resulting cultures were grown at 37 °C with shaking at 175 rpm in 4 L baffled flasks. Protein expression was induced by addition of IPTG (0.5 mL of a 0.5 M aqueous stock solution, 250 μM final concentration) at OD600 ~ 0.6 (t ~ 5.5 h). The cultures were incubated at 37 °C with shaking at 175 rpm for an additional 4 h, and the cells were immediately pelleted by centrifugation (4,000 rpm × 30 min, 4 °C). 15N-labeled HD5ox was overexpressed on a 4-L scale and the 15N-labeled mutant peptides were each overexpressed on a 12-L scale. The final OD600 values varied from ca. 0.7 to ca. 1.2 depending on the shaker flask. The resulting cell pellets were collected, flash frozen in liquid N2, and stored at -80 °C. The wet pellet yield for N-His6-Met-HD5 was ca. 2 g/L culture. Wet pellet yields of ca. 1.2 and ca.1.8 g/L culture were obtained for 15N-His6-Met-HD5[Ser] and 15NHis6-HD5[Ser], respectively. Overexpression of double-labeled C,N-His6-Met-HD5 was performed on a 6-L scale by using the same method and substituting U-13C-glucose for unlabeled glucose. Isotopically-labeled His6-HD5 and the His-tagged mutant peptides were purified as described previously for the unlabeled congeners.12 In brief, the His6-tagged HD5 and serine double mutants were isolated in yields of ca. 5-15 mg/L culture following Ni-NTA affinity chromatography. Each His6 tag was cleaved by using cyanogen bromide, and each crude peptide reduced by addition of TCEP and HPLC purified. An oxidative folding procedure was employed to obtain the oxidized forms, which were separated and purified by semipreparative HPLC.12 Peptide purity was ascertained by analytical HPLC (Figures S1-S4), and peptide identities were confirmed by mass spectrometry (Table S2). The purified peptides were lyophilized to dryness and stored as powders at −20 °C until use. Some disulfide bond shuffling was observed by analytical HPLC for select unlabeled disulfide deletion mutants after several months of storage at -20 °C in neutral aqueous solution. As a result, the 15N-labeled disulfide regioisomers of the serine double mutants were stored as lyophilized powders until use, and characterized immediately following purification. Wommack et al. Page 4 Biochemistry. Author manuscript; available in PMC 2013 December 04. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript Peptide Stability in the Presence of Staphylococcus aureus S. aureus ATCC 25923 was grown overnight with shaking (37 °C, 16 h) in 5 mL of TSB. The overnight culture was diluted 1:100 into 6 mL of fresh TSB and grown for ~2 h at 37 °C with shaking at 150 rpm until the OD650 reached ~0.6. A 5-mL portion of the culture was transferred to a sterile culture tube and centrifuged (3500 rpm × 10 min, 4 °C) to pellet the bacterial cells. The supernatant was discarded and the cell pellet was resuspended in 5 mL of AMA buffer (10 mM sodium phosphate buffer supplemented with 1% TSB, pH 7.4). The cell suspension was centrifuged (3500 rpm × 10 min, 4 °C) and the supernatant discarded. The resulting cell pellet was resuspended in 5 mL of AMA buffer and diluted with AMA buffer to obtain an OD650 value of 0.6 (1 × 108 CFU/mL). This bacterial suspension was further diluted 1:100 in two steps (1:10 × 1:10) into 2 mL of AMA buffer. The diluted cultures were used immediately. Peptide stability assays were performed in 96-well plates. Each well contained 10 μL of a 200-μM (10x) aqueous sterile-filtered peptide stock solution or a no-peptide control. A 90μL aliquot of the diluted bacterial culture was added to each well and the plate was incubated for 1 h (37 °C, 150 rpm). Wells containing AMA buffer only and peptide in the AMA buffer without S. aureus were also included. Immediately after the 1 h incubation, each culture was transferred to a microcentrifuge tube and the samples were centrifuged (13,000 rpm × 10 min, 4 °C). The supernatants were transferred to new microcentrifuge tubes, a 10-μL aliquot of 2% aqueous TFA was added to each solution, and the samples were centrifuged (13,000 rpm × 10 min, 4 °C). The resulting supernatants were transferred to HPLC vials and stored in an autosampler thermostated at 4 °C until analytical HPLC analysis (10-60% B over 30 min). This assay was conducted at least in triplicate for each peptide and over two separate days. Representative HPLC traces are reported in Figures 2 and S5-S6. Solution NMR Sample Preparation Samples of N-HD5ox were prepared at different concentrations and pH values to determine the optimal sample conditions for NMR data collection. Initial data acquisition was performed on a 460-μM sample of N-HD5ox that was dissolved in 90:10 H2O/D2O immediately after HPLC purification and lyophilization (Figure 3). Additional samples of N-HD5ox were prepared at pH 5.0 (630, 460, and 260 μM) by using an aqueous solution of 1 N HCl for adjusting the sample pH. In a separate screen, N-HD5ox samples at pH 7.0 (333 μM), 6.0 (340 μM), 5.0 (400 μM), and 4.0 (460 μM) in 90:10 H2O/D2O were prepared by using TFA to adjust pH as necessary. To determine the effect of buffer, samples of N-HD5ox (800 μM) were prepared in 20 mM Tris-HCl buffer containing 10% D2O (v/v) at pH = 7.0, 6.0, and 5.0. Lastly, N-HD5ox (880 μM) was prepared in 10 mM sodium phosphate buffer with 10% D2O (v/v) at pH = 7.0, 6.0, and 4.0. In these two sets of samples, the sample pH was adjusted by incremental additions of 1N HCl. Based on the 1H,15N-HSQC spectra of N-HD5ox prepared under various conditions, the 13C,15NHD5ox sample (340 μM) was prepared in 90:10 H2O/D2O at pH 4, and TFA was employed to adjust the sample pH. These conditions afforded the greatest peak dispersion, and twentyeight of thirty-one amide resonances were observed for C,N-HD5ox in the 1H,15NHSQC. Likewise, all N-HD5[Ser]ox and N-HD5[Ser]ox regioisomers were prepared in 90:10 H2O/D2O at pH 4. The NMR sample of N-HD5red (650 μM) was prepared in 90:10 H2O/D2O containing 20 μM TFA to ensure that the peptide remained reduced. Solution NMR Spectroscopic Studies All 1-D 1H NMR spectra were collected on a Varian 500 MHz spectrometer housed in the MIT Department of Chemistry Instrumentation Facility (DCIF) that was operated at an Wommack et al. Page 5 Biochemistry. Author manuscript; available in PMC 2013 December 04. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript ambient probe temperature of 293 K (Figures S7-S8). Standard techniques for water suppression and data acquisition were employed. A number of multi-dimensional NMR spectra were recorded on a 600 MHz NMR spectrometer housed in the MIT Francis Bitter Magnet Laboratory (FMBL) based on a FBML narrow bore magnet and a console designed and constructed by members of the FBML. This spectrometer is equipped with three transmitter channels, and a Nalorac 5 mm indirect triple resonance 1H[13C,15N] probe with z-gradient. Additional multi-dimensional NMR spectra were recorded on a 600 MHz Bruker Avance spectrometer equipped with a cryogenic probe housed at Harvard Medical School. To determine optimal acquisition conditions for HD5ox , 1H,15 N-HSQC experiments were performed at 15 °C, 20 °C, and 25 °C. For the initial resonance assignments, TOCSY and NOESY experiments were performed at 25 °C. 2-D TOCSY spectra were recorded with mixing times of 30 and 60 ms, and 2-D NOESY spectra were recorded with mixing times of 150, 200, and 400 ms. All experiments were acquired with 2048 complex points in t2 and 512 complex points in t1, and a sweep width of 12 ppm in both dimensions. The 3-D 15Nedited TOCSY and 3-D 15N-edited NOESY experiments were collected with 60 ms and 200 ms mixing times, respectively. A 200 ms mixing time was also employed for a 3-D 13Cedited NOESY experiment. Sequence-specific assignment was aided by the collection of standard HNCA, HNCO, and HNCACO pulse sequences; however, non-uniform sampling was used. Specifically, a matrix of 38 points (15N dimension) by 40 points (13C dimension) at the ca. 20% levels (a total of 320 acquired complex points) was sub-sampled. The sampling schedule was created based on the Poisson Gap sampling method.48 Missing data points were reconstructed by using the istHMS algorithm.49 Only 1-D 1H NMR and 2-D 1H,15N-HSQC spectra for the HD5[Ser]ox and HD5[Ser]ox regioisomers were collected, and the HSQC experiments were conducted over a temperature range of 15 to 25 °C. Spectral data were processed by using NMRPipe50 and analyzed by using Sparky51 or CARA.52 NMR Solution Structure Calculations and Refinement Structure calculations were initially performed in CYANA to fully assign NOE crosspeaks and establish the hydrogen bond network by inference from preliminary structures along with NOE patterns. These NOE assignments were then used in structure calculations with X-PLOR NIH using explicit water refinement. During this calculation, the system was cooled from 3000 to 25 K within 10 psec, applying the high force constants obtained at the end of the previous cooling stage. The experimental restraints included 421 upper distance limits, fifty-four dihedral angles identified by analysis of backbone chemical shifts by the program TALOS,53 sixteen X1 angles, three disulfide bonds, and fifteen hydrogen bonds. Of the 400 structures resulting from the final round of structure calculation, the twenty lowest-energy structures were selected. The geometry and elements of secondary structure were analyzed using PROCHECK.54 These coordinates are deposited in the Protein Data Bank (code: 2LXZ). The UCSF Chimera55 package and MOLMOL56 were employed for final graphical presentation. Sedimentation Velocity Experiments A Beckman XL-I Analytical Ultracentrifuge outfitted with an An-50 Ti rotor was employed for all sedimentation velocity (SV) experiments. The rotor housed conventional doublesector charcoal-filled epon centerpieces within the sample cells and contained either sapphire (Rayleigh interference optics) or quartz (absorption optics) windows. The absorption wavelength for optical detection was 280 nm and the interferometer laser wavelength was 660 nm. The samples were centrifuged at 42,000 rpm and 20 °C until sedimentation was complete. SEDNTERP57 was employed to calculate the buffer viscosity (η), buffer density (ρ), and protein partial specific volume (v-bar) values at 20 °C based on a Wommack et al. Page 6 Biochemistry. Author manuscript; available in PMC 2013 December 04. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript database of known values available via the Internet (http://www.jphilo.mailway.com). The sedimentation coefficients were subsequently calculated by fitting the sedimentation velocity data using SEDFIT. The continuous distribution c(s) Lamm equation model, which accounts for protein diffusion, was employed.58 The sedimentation coefficients generated by this approach were confirmed by using DCDT+.50,60 The apparent sedimentation coefficient distribution, g(s*), was generated from 22-26 scans with a peak broadening limit of 60 kDa using DCDT+. All SV window assemblies were loaded with 410 μL of buffer reference and 400 μL of peptide sample, and the buffers and samples were prepared immediately before the SV runs. In one set of experiments, samples of HD5ox, the HD5[Ser]ox and HD5[Ser]ox regioisomers, and HD5[Serhexa] were prepared at pH 7 in 10 mM sodium phosphate buffer. A solution of 1N HCl was employed to adjust pH. Starting from a lyophilized peptide sample, a concentrated stock solution of each peptide was prepared from buffer that was filtered through a 0.45 μm membrane. In microcentrifuge tubes, aliquots of the peptide stock solution were diluted to 400 μL with buffer to provide the desired concentrations and subsequently transferred to the AUC sample cells. Samples at the following peptide concentrations were prepared and analyzed: HD5ox, 30, 50, 80, 115, 120, 183, 186, 283, 301, 303, 424, and 437 μM; HD5[Ser]ox (5-20)(10-30), 60, 62, 65, 90, and 131 μM; HD5[Ser]ox (5-30)(10-20), 105, 136, and 201 μM; HD5[Ser]ox (5-10)(20-30), 74, 105, 153, and 210 μM; HD5[Ser]ox (3-20)(5-31), 153, 180, 224, and 236 μM; HD5[Ser]ox (3-31)(5-20), 57, 232, and 396 μM; HD5[Serhexa], 90 and 91μM. Additional SV experiments were conducted to evaluate the consequences of pH, salt, and buffer components on the sedimentation of HD5ox. In all cases, the 400-μL solutions were prepared as described above and the buffer pH was adjusted by using 1 N HCl. To determine the effect of pH, samples of HD5ox in 10 mM sodium phosphate buffer were adjusted to pH values of 6 (161 μM), 4 (131 μM), and 2 (194 μM). To ascertain the effect of NaCl, samples of HD5ox at pH 7 in 10 mM sodium phosphate buffer containing 50 mM (183, 283 μM), 150 mM (181, 278 μM), and 500 mM (178, 270 μM) NaCl were prepared. To evaluate the effects of buffer choice and divalent cations, sedimentation of HD5ox was investigated at pH 7 in 20 mM Tris-HCl or 20 mM HEPES buffer with or without 50 mM MgCl2 or CaCl2. For the experiments in Tris buffer, the HD5ox concentrations were 126 and 210 μM (no divalent cations), 170 and 236 μM (+Mg), or 128 and 157 μM (+Ca). For the experiments in HEPES buffer, the HD5ox concentrations were 191 and 256 μM (no divalent cations), 131 and 190 μM (+Mg), and 191 and 212 μM (+Ca). Hydrodynamic modeling computations were performed with HYDROPRO61 to calculate sedimentation coefficients for the HD5ox monomer, dimer, and tetramer (Table S3). Both the HD5ox monomer NMR solution structure presented in this work and the reported HD5ox crystal structure (PDB: 1ZMP)10 were employed in hydrodynamic modeling. All HYDROPRO calculations used the buffer density (ρ) and buffer viscosity (η) values for water at 20 °C, and a partial specific volume (v-bar) of 0.7087 mL/g for HD5ox. Equation 1 was employed to calculate sedimentation coefficients for HD5ox modeled as a smooth, compact, and spherical peptide in water at 20 °C using the classical combination of the Svedberg and Stokes equation.58 The values are reported in Tables S4. Equation 1 states