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Review
. 2016 Jul 5:45:135-52.
doi: 10.1146/annurev-biophys-062215-011121. Epub 2016 Apr 27.

Protein Folding-How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry

S Walter Englander  1 , Leland Mayne  1 , Zhong-Yuan Kan  1 , Wenbing Hu  1
Affiliations
Review

Protein Folding-How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry

S Walter Englander et al. Annu Rev Biophys. .

Abstract

Advanced hydrogen exchange (HX) methodology can now determine the structure of protein folding intermediates and their progression in folding pathways. Key developments over time include the HX pulse labeling method with nuclear magnetic resonance analysis, the fragment separation method, the addition to it of mass spectrometric (MS) analysis, and recent improvements in the HX MS technique and data analysis. Also, the discovery of protein foldons and their role supplies an essential interpretive link. Recent work using HX pulse labeling with MS analysis finds that a number of proteins fold by stepping through a reproducible sequence of native-like intermediates in an ordered pathway. The stepwise nature of the pathway is dictated by the cooperative foldon unit construction of the protein. The pathway order is determined by a sequential stabilization principle; prior native-like structure guides the formation of adjacent native-like structure. This view does not match the funneled energy landscape paradigm of a very large number of folding tracks, which was framed before foldons were known and is more appropriate for the unguided residue-level search to surmount an initial kinetic barrier rather than for the overall unfolded-state to native-state folding pathway.

Keywords: HX MS; energy landscape theory; hydrogen exchange; protein folding.

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Figures

Figure 1
Figure 1
The defined pathway view: a distinct folding pathway through distinct kinetic intermediates. The model implies the existence of a ladder of defined intermediates at equilibrium in the high free-energy space above N (right). The colors represent the set of five cytochrome c foldons common to the native protein (lower left), the equilibrium energy ladder of partially unfolded forms (right), and the kinetic folding pathway. The cooperative foldon units that compose native cytochrome c were first defined by native state hydrogen exchange (HX) studies (see Figure 3) (5). The corresponding ladder of partially unfolded forms was defined by stability labeling HX experiments (40, 41). The kinetic pathway is considered here and in other publications (27, 62). Abbreviations: U, unfolded state; TS, transition state; N, native state.
Figure 2
Figure 2
Amide hydrogen exchange (HX) chemistry. Main-chain amide HX is catalyzed by hydroxide ion (right limb) and hydrogen ion (left limb). The HX rate of any given amide hydrogen is modulated also by its two nearest neighbor side chains. The different V-shaped curves illustrate some alternative neighbor combinations (3).
Figure 3
Figure 3
Native state hydrogen exchange (HX) results for cytochrome c distinguish subglobal foldon unfolding reactions (high denaturant dependence) from local fluctuational HX (zero denaturant dependence) (2, 5). Unfolding free energy was computed from HX rates measured by 2D nuclear magnetic resonance as a function of low levels of denaturant (Equation 3) and extrapolated to zero denaturant. The different panels show the residues that identified the (a, b) blue and (c) green foldons, and less definitively the (d) yellow and red foldons. The lower-lying foldons were better defined in later work.
Figure 4
Figure 4
Hydrogen exchange (HX) mass spectrometry (MS) pulse labeling experiments with apoflavodoxin (Z-Y Kan, W.K. Lim, L. Mayne, S.W. Englander, unpublished data). (a) Experimental setup. (b) Peptide fragments obtained. Note that the high degree of peptide overlap provides many internal consistency checks, both in verification of peptide identity and in HX data checking. Also, any given residue is presented redundantly in many different peptide-overlapping regions and peptide overhangs, promoting the ability of the HDsite analysis (31) to obtain near-single residue resolution. (c) The time evolution of three illustrative peptide envelopes. (d) Kinetic folding seen for many peptides distinguishes five different folding units; a sixth group remains unprotected in the native protein. The peptides shown in black and red exhibit back unfolding during the labeling pulse, which transfers amplitude from the heavier to the lighter envelope (EX1 HX), and independently demonstrates their separately cooperative unfolding nature. Abbreviations: DL, delay line; ESI-MS, electrospray ionization mass spectrometry; HPLC, high-performance liquid chromatography.
Figure 5
Figure 5
Hydrogen exchange mass spectrometry pulse labeling results for peptides from (a, b) RNase H (27), (c) MBP (62), (d) cyt c (W. Hu, Z-Y Kan, L. Mayne, S.W. Englander, unpublished data), and (e) CRABP P85A (W. Hu, Z-Y Kan, L. Mayne L. Gierasch, A.K. Thakur, S.J. Eyles & S.W. Englander, unpublished data). The energy ladder of states follows the same color coding as above, arranged in spectral order of increasing energy. The black curves for MBP trace the late-stage folding of sites that showed significant protection in the initial prefolding protein collapse. Removal of the single CRABP proline (P85A) makes folding appear to be two-state. For all proteins, the colors show the time course for folding of the blue, green, yellow, red, and infrared foldons, named in spectroscopic order of decreasing unfolding free energy. Abbreviations: CRABP, cellular retinoic acid binding protein; cyt c, cytochrome c; MBP, maltose binding protein; RNase H, ribonuclease H.
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References

    1. Anfinsen CB, Haber E, Sela M, White FH., Jr The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. PNAS. 1961;47:1309–14. - PMC - PubMed
    1. Bai Y, Englander SW. Future directions in folding: the multi-state nature of protein structure. Proteins. 1996;24:145–51. - PubMed
    1. Bai Y, Milne JS, Mayne L, Englander SW. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993;17:75–86. - PMC - PubMed
    1. Bai Y, Milne JS, Mayne L, Englander SW. Protein stability parameters measured by hydrogen exchange. Proteins. 1994;20:4–14. - PubMed
    1. Bai Y, Sosnick TR, Mayne L, Englander SW. Protein folding intermediates: native-state hydrogen exchange. Science. 1995;269:192–97. - PMC - PubMed

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