Figure 1.6.: Schematic model of the domain structure of Zuo~49 kDa, 433 aa
Although several studies support the function of RAC/Ssb as a chaperone machine, the actual role of this system in the process ofde novoprotein folding is still unresolved.
It is unknown how the chaperone system is targeted to the ribosome and how it acts on nascent polypeptides on the ribosomal surface. One aspect which is of decisive importance for the functionality of the triad is the mode of assembly of Ssz and Zuo into the RAC heterodimer, for proper interaction with Ssb. It also remains unclear whether the nucleotide ATP plays a role in this binding/association, and what are the particular dynamics of the protein-protein interaction of Zuo with Ssz.
1.7. Analysis of Protein Dynamics
To monitor conformational properties of proteins on a global level, a variety of methods are available such as far-UV circular dichroism (CD) (Pelton and McLean, 2000), tryptophan fluorescence, infrared spectroscopy (Barth, 2000) as well as small-angle X-ray scattering and cryo-electron microscopy (Koch et al., 2003).
In order to determine detailed information about amino acid structures within proteins, X-ray crystallography is capable of determining resolution in the range of 1-2 Å(Moffat, 2001). However, this can only be achieved using highly ordered crystallized protein.
Since a long time, attempts were made to crystallize RAC, Zuo and Ssz, respectively, but so far no crystals could be obtained.
Since many years multi-dimensional nuclear magnetic resonance (NMR) spectros-copy is used to monitor protein dynamics in solution. This is possible under steady state conditions and on multiple time scales at the amino acid level (Dyson and Wright, 2004). However, NMR experiments have long been limited to small, soluble proteins up to 35 kDa. New technologies (high magnetic fields and cryoprobes) and new pulse sequences now allow to analyze proteins up to 100 kDa and possibly even very large structures such as ribosomes, depending on the system used and the biological question
to answer (Henzler-Wildman and Kern, 2007).
Determination of global or local dynamics within a protein can also be done by hydrogen exchange (HX), which can be coupled to either mass spectrometry or NMR spectroscopy. It provides a powerful tool for analysis on a high temporal resolution, reaching from the millisecond range up to hours. A method to detect local flexibility of distinct atoms within molecules is electron paramagnetic resonance (EPR). Mass spectrometry, HX measurements and EPR were the methodology used in this work to investigate conformational changes within chaperone proteins. Details of mass spectrometry, HX measurements and EPR can be found in the appendix. A short introduction into HX-MS and the setup used in this study will be described in the following section.
1.7.1. Amide hydrogen exchange and mass spectrometry (HX-MS) Mass spectrometry has proven to be a valuable analytical tool to measure amide hy-drogen exchange to monitor protein dynamics and conformational changes (Hoofnagle et al., 2003; Kaltashov and Eyles, 2002; Wales and Engen, 2006). A major advantage of mass spectrometry is that it separates coexisting conformational states of a protein by their differences in deuterium contents and hence molecular masses. This feature of mass spectrometry is unique as other techniques, such as e.g. NMR which is also used to analyze amide hydrogen exchange, only measure average behavior of different con-formational states. Additional advantages of mass spectrometry are the lower amount of sample, required for one run, the unlimited mass range for larger proteins and the ability to monitor individual peptides and proteins in a mixture. Due to its coupling to HPLC, ESI-MS is the most commonly used ionization method in HX studies.
In a typical HX experiment, undeuterated proteins are labeled with deuterium by dilu-tion in a D2O buffer. Incubating for different time intervals in D2O and monitoring the isotope exchange as a function of exchange time provides information on the conforma-tional dynamics of a protein under equilibrium conditions. In an established labeling protocol (Zhang and Smith, 1993) the exchange reaction is performed at physiological conditions for certain amounts of time and subsequently quenched by lowering the pH to 2.5 and the temperature to 0◦C, which decreases HX rates at the peptide amide linkages by up to five orders of magnitude. The half-life for amide hydrogen exchange under these quench conditions is 30 - 120 min. This allows to analyze the sample by
1.7. Analysis of Protein Dynamics reversed phase HPLC coupled to ESI-MS, causing minimal loss of deuterons on the
amides.
Figure 1.7.: Experimental setup of an amide hydrogen exchange experiment com-bined with mass spectrometryThe protein is equilibrated under physiological conditions and then diluted into D2O buffer for certain time intervals (tHX). After a quench at low pH and low temperature, the deuterated protein is injected into a HPLC for desalting and sub-jected to ESI-MS. Measurement of deuteron incorporation in full-length protein mass spectra results in information about global exchange kinetics. In addition, the deuterated protein can be digested during the HPLC step by pepsin and the deuteration kinetics of the peptic pep-tides (i.e. shifts of the isotopic cluster to higher m/z by deuteration time) provides information about slow and fast exchanging regions in the protein.
To determine the exchange behavior of a protein on a global level, deuteron incorpo-ration of full-length mass can be determined. To obtain information of regions in the protein that exchange either slowly or rapidly, a proteolytic digest of the deuterated protein and MS analysis of the resulting peptides can be accomplished. The most commonly used acidic protease is pepsin. Pepsin provides peptides with an average length between 4-15 amino acids. A disadvantage of this procedure is a continuous loss of deuterium label during HPLC analysis in aqueous buffers. This effect called
"back exchange" can be reduced by fast sample processing at low temperatures (Feng et al., 2006).