Structural basis of protein stability at poly extreme crystal structure of amya at 1 6 a resolution 3

The stability of a native protein structure at different conditions can be assessed by measuring the change in the Gibbs free energy, ΔG, forunfolding by temperature or denaturant, as mo

CHAPTER 3 BIOPHYSICAL CHARACTERIZATION OF AmyA

3.1 INTRODUCTION

Biophysical approaches, which are complementary to detailed molecular protein structures from crystallography, can provide data that reveal insights into how proteinsbehave in solution and how they interact dynamically with eachother From X-raystudies one may get three-dimensionalstructures with different ligands bound

or structures at different conditions From solution spectroscopy one may get a continuous variation of distribution of structures between several conditions, e.g

ligand binding, pH, temperature and salt concentration The complete understanding

of the shape, size and thermal stability of proteins requires different biophysical approaches

Classical biophysicalanalyses to determine size, shape, and solution properties

of proteins have been around since the time of viscosity measurements and sedimentation analysis using the analytical ultracentrifuge The size and shape of molecules can be estimated by monitoringthe movement of these molecules in a force field such as centrifugal force for sedimentation, gravity for gel filtration, and electricalpotential for electrophoresis

The stability of a native protein structure at different conditions can be assessed by measuring the change in the Gibbs free energy, ΔG, forunfolding by temperature or denaturant, as monitored by UV or florescence spectroscopy A combination of these methods is frequently the most effectivemeans to characterize proteins Since AmyA is active and stable in a wide range of salt concentration at high temperature, it is important to know how AmyA behaves in solution at different salt

concentration To understand the mechanisms by which AmyA in solution handles

two extreme conditions we carried out a series of biophysical experiments

3.2.1 Circular dichroism (CD) measurements

CD measurements were made with a Jasco J-715 spectropolarimeter equipped with a Peltier cell holder and a PTC-348WI temperature controller A cuvette with 0.1

cm pathlength was used throughout A protein concentration of 0.1 mg ml-1 (determined by the Bradford assay method) was used for wavelength scans For wavelength scans, a scan rate of 10 nm min-1 was used with a time constant of 4 s and

3 scans were averaged Data were collected at 65 ºC over a wavelength range of

190-260 nm with a bandwidth of 1 nm The solvent spectrum was subtracted from the sample spectrum The far UV CD spectra were analyzed by the secondary structure

analysis program CDNN, version 2.1 (Böhm et al, 1992) and data from 190-210 nm

were excluded from analysis as NaCl is known to have signals in this region of the spectrum Thermal melting was carried out at a scan rate of 5 °C per minute at 222 nm using protein samples at different salt concentrations in 50 mM Tris (pH 8.0) and heated from 40 to 100 ºC

3.2.2 Analytical ultracentrifugation

Sedimentation velocity experiments were performed with a Beckman Optima XL-I at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies (CAUMA, University of Texas Health Science Center, San Antonio, USA) All samples were analyzed in 50 mM Tris buffer (pH 8.0) containing varying amounts of

speeds ranging between 3,000-50,000 rpm Absorbance samples were spun in 2-channel epon/charcoal center-pieces in the AN-50-TI or AN-60-TI rotor Scans were collected at 280 nm and using interference optics in the continuous mode with 0.003

cm step size setting and no averaging Loading concentrations ranging between 0.9-1.2 OD were measured at the given wavelength

Data were analyzed with UltraScan, version 6.2 (http://www.ultrascan.uthscsa.edu) Hydrodynamic corrections for buffer conditions

were applied according to Laue (Laue et al, 1992), and as implemented in UltraScan

The partial specific volume of AmyA was estimated according to the method by Cohn and Edsall (Cohn and Edsall, 1943), and as implemented in UltraScan Data were

analyzed using the van Holde–Weischet (van Holde et al, 1978) method, which

reports sedimentation coefficient distributions for the sample Sedimentation coefficient distributions were transformed into molecular weight distributions by applying the Svedberg equation and assuming a particle shape that corresponds to an

expected frictional ratio, f/f 0

3.2.3 Analytical gel filtration

AmyA was subjected to gel filtration chromatography using an analytical Superdex-75 column (Amersham Pharmacia) on a Duo Flow FPLC system (Bio-Rad) The column was equilibrated with respective NaCl concentration containing 50 mM Tris (pH8.0) AmyA samples at different NaCl concentrations were loaded to the column and eluted at a flow rate of 0.5 ml min-1

3.3 RESULTS

3.3.1 Thermal stability at different salt concentrations

To understand the relationship between the presence of salt and the thermal stability of AmyA, temperature melt experiments were carried out at different salt concentrations Denaturation of AmyA was monitored by circular dichroism The thermal stability of AmyA increases with respect to the increase in NaCl concentration (Fig 3.1) An increase of 7 °C in Tm was observed at 4.7 M NaCl concentration compared to 100 mM NaCl concentration The thermal stability of AmyA remains the same when NaCl was replaced with other similar monovalent salts like KCl, NaBr, RbCl and CsCl (data not shown) However, the addition of the bivalent salt CaCl2 significantly increased the thermal stability of AmyA At high NaCl concentration, however, the presence of CaCl2 did not show any marked difference in the Tm

Figure 3.1 The CD spectra of AmyA at different salt concentration

The CD spectra monitored at 222 nm demonstrating the thermal

On the other hand, we observed most surprisingly that AmyA showed the maximum thermal stability in the complete absence of any salt The protein was extremely stable up to 100 °C when there was no salt Furthermore, AmyA that was pre-incubated for 30 minutes in the complete absence of NaCl in boiling water still retained its activity (Fig 3.2)

Figure 3.2 Activity of AmyA after temperature melt Activity assay

of AmyA at different NaCl concentration before and after the

incubation of the AmyA sample in boiling water for 30 minutes

Addition of as low as 5 mM NaCl or CaCl2 to this solution decreased the thermal stability and the melt was observed at 85 °C To understand the conformational changes at these conditions, the Far-UV CD spectrum of AmyA, both

in the presence and absence of salt, was analyzed (Fig 3.3) Surprisingly, no significant secondary structural change was detected This indicates that AmyA retains the overall fold at the entire salinity as seen in the low salt (lAmyA) and high salt (hAmyA) crystal structures

Figure 3.3 The Far-UV CD spectra of AmyA at different NaCl concentration

3.3.2 Novel oligomerization and its implications for stability and function

Several studies have shown that many halophilic proteins are involved in salt

dependent oligomerization (Ishibashi et al, 2002; Jekow et al, 1999) To determine

whether AmyA undergoes any salt dependent oligomerization, we analyzed the quaternary structure of AmyA by analytical ultracentrifugation and gel filtration chromatography The sedimentation behavior of the AmyA protein showed a very strong dependence on NaCl concentration When no salt was present, the protein had

a strong tendency to oligomerize and formed very large aggregates (Fig 3.4)

Sedimentation distributions were compared with the van Holde-Weischet analysis, which provides model independent sedimentation coefficient distributions

that are corrected for diffusional boundary spreading (Demeler et al, 1997; Demeler et

al, 2004) By running at a relatively low speed (3000 rpm) it was possible to measure

an S-value distribution of the sample which indicated that 70% of the protein was

between 20-80 S, and the remainder at larger S-values This corresponds to aggregates with a molecular weight distribution between 4-5.5 million Dalton with the majority

of the sample around 5 million Dalton

Figure 3.4 Molecular weight distribution of AmyA at no salt The

peak corresponds to a poly-dispersed aggregate of AmyA

However, these aggregates retain 40% of activity when tested (Fig 3.2) Addition of as low as 5 mM of NaCl reversed most of the protein aggregates into the

monomeric form, sedimenting between 3.5 - 4.5 S, corresponding to the molecular

weight of the monomer (Fig 3.5) The molecular weight and frictional coefficient ratios were further confirmed by fitting the data from samples containing salt with

finite element solutions of the Lamm equation (Cao et al, 2005) (data not shown)

which confirmed the monomeric state for all samples containing salt

Figure 3.5 Molecular weight distributions of AmyA in different

NaCl concentrations The molecular weight of the peaks corresponds

to that of an AmyA monomer which is 55 Kilo Dalton

Additionally, we confirmed this behavior with gel filtration chromatography Results from gel filtration chromatography with a Superdex-75 column at room

temperature are consistent with the results from analytical ultracentrifugation In the

absence of NaCl, at 50 mM Tris (pH 7.5) AmyA exists in a large sized oligomeric state (Fig 3.6) Inter-conversion between the monomer and oligomer is strongly influenced by salt concentration Both the experiments indicate that in the absence of salt AmyA forms a poly-dispersed oligomeric state without losing its tertiary structure and activity

Figure 3.6 Gel filtration profile of AmyA at different NaCl concentrations

Oligomeric proteins are more thermostable than their monomers due to inter-subunit interactions and often thermophilic and halophilic proteins form oligomers

(Ishibashi et al, 2002; Jekow et al, 1999; Richard et al, 2000) This indicates that

inter-subunit contacts of AmyA at the oligomeric state could be the reason why it is very stable in the absence of salt Also, this result suggests that AmyA oligomerization might occur mainly through the high affinity ion binding sites that are present on the surface Addition of salt promotes salt binding to those sites and removes the inter-subunit interactions This oligomerization is very different from the previously observed oligomerization in halophilic proteins in which oligomers are formed at high salt concentrations due to the increase in water surface tension (Lin and Timasheff, 1996) At low salt concentration these oligomers dissociate into monomers and the protein loses its stability