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Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation Ke Xia, Sandy Pittelli, Jennifer Church, and Wilfredo Colon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01965 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 25, 2016

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Journal of Agricultural and Food Chemistry

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Kinetic Stability of Proteins in Beans and Peas:

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Implications

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Germination, and Plant Adaptation

for

Protein

Digestibility,

Seed

4 5 6

Ke Xia, Sandy Pittelli, Jennifer Church, Wilfredo Colón*

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Department of Chemistry and Chemical Biology, Center for Biotechnology

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and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New

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York 12180

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Correspondence and requests for materials should be addressed to W.C.

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(e-mail: [email protected]).

ACS Paragon Plus Environment


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ABSTRACT: Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium

15

dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and

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may have arisen in nature as a mechanism for organismal adaptation and survival against harsh

17

conditions. Legumes are well known for possessing degradation-resistant proteins that often

18

diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-PAGE, a

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method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes

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(mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show

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beans that are more difficult to digest, such as soybean, Lima beans, and various common

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beans, have high content of KSPs. In contrast, mung bean, red lentil, and various peas that are

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highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are

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associated with warm season beans, which germinate at higher temperatures. In contrast, peas

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and red lentil, which are cool season legumes, contain low levels of KSPs. Thus, our results

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show protein kinetic stability is an important factor in the digestibility of legume proteins, and

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may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic

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stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied

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for determining the abundance and identity of KSPs in engineered and wild legumes, and for

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advancing basic research and associated applications.

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Keywords: SDS-resistance, hyperstability, protease-resistance, proteomics,

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INTRODUCTION

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Among plants of human consumption, legumes contain the highest amount of proteins. In

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particular, beans and peas are a major source of protein nutrition. The common bean

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(Phaseolus vulgaris L.) is consumed all over the world, especially in Latin America and Africa.

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Soybeans are a globally important crop, providing oil and protein for human and animal

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consumption. A major concern of soybean and common beans has been the presence of

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biochemical factors limiting the nutritional value of their proteins. Among the most important

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antinutritional factor is the limited digestibility of certain proteins, largely due to the structural

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properties of these proteins.1 Because of their importance in human nutrition and agriculture,

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legume proteins have been the subjects of many research studies to understand the basis of

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their digestibility and antinutritional factors. The biological roles of these proteins have also been

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of much interest. In beans and peas, highly abundant globulin-like proteins serve as storage

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proteins to provide a source of amino acids during plant germination, and they comprise up to

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70% of the proteins in these legumes. In some legumes, highly abundant proteins may also

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serve to protect them against insects.2

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Among storage proteins in dry beans, phaseolins are known to be more resistant to

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proteolytic digestion, whereas storage proteins in peas (i.e. vicilin) are more digestible.3 Glycinin

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and P-conglycinin proteins in soybean have intermediate susceptibility to various proteases.3

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From a nutrition standpoint, the stability of storage proteins has implications for human

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digestibility and nutritional value.4 The relationship between proteolytic resistance of unheated

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legume proteins and their poor nutritional value is well known.5 Furthermore, some bean

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proteins have been linked to allergy and digestive intolerance.6 Allergy to soy and beans is

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relatively common in the general population.7 In animals, pre-ruminant calves suffer from a

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gastrointestinal hypersensitive response to certain soybean products due to the inability of their

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digestive tract to denature antigenic constituents of the soybean protein.8 Soy and common



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beans may also trigger digestive symptoms due to food intolerance. The structure and

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degradation-resistance of certain legume proteins are known to play a major role in their

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digestive intolerance and allergenic potential.9

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Although protein structure and stability are known contributors, the biophysical basis for the

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degradability differences of storage protein is not clearly understood. N-terminal amino acids (i.e.

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N-end rule) are known to contribute significantly to a protein’s half-life, but protein turnover is

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also affected by protein-protein interactions, physical location of the protein, presence of

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cofactors, and post-translational modification (e.g. glycosylation).10 In addition, the structural

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properties of the proteins themselves, which contribute to Kinetic Stability (KS), are likely key

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factors in their digestibility.5 This is consistent with the observation that a protein’s protease

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resistance is mostly related to its KS, rather than its thermodynamic stability (TS).11 The two

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types of protein stability are illustrated in Figure 1. Kinetic stability is a term used to describe

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hyperstable proteins that are conformationally trapped in the native state by a high energy-

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barrier, and thereby protected from degradation resulting in a longer half-life.12 In contrast,

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protein TS is the difference in Gibbs free energy between the native and unfolded state, and

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these states usually exist in equilibrium with each other that favors the native state. The stability

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of most proteins is under thermodynamic control, making them susceptible to proteolytic

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degradation due to the exposure of cleavage sites during their transient unfolding. Thus, the

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height of the unfolding barrier will largely determine a protein’s protease susceptibility.

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Kinetic stability appears to be a strategy used by nature to protect organisms against harsh

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conditions.12 To gain insight about the biological, agricultural, and nutritional implications of

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KSPs in legumes, we applied the method of diagonal two-dimensional (D2D) SDS-PAGE, which

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allows the separation of KSPs from other proteins in complex biological extracts. Mass

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spectrometry and proteomics analysis was then used to determine the abundance and identity

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of KSPs in beans and peas. Our results identify KS as a key biophysical factor in the digestibility


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of legume proteins, and have implications for further understanding of the nutritional efficiency

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of legumes and the biological roles of protein KS in legume germination and adaptation.

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MATERIALS AND METHODS

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Sample Source and Preparation. In order to test the most representative samples from

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public food source, dry black eye pea, navy bean, kidney bean, small red bean, Lima bean, split

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pea, chick pea, black bean and pigeon pea were purchased from local supermarkets, including

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Walmart and Price Chopper stores. Dry soybean, lentils, mung bean and adzuki bean were

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purchased from Whole Foods. 10 g of dry beans were mixed with 40 mL extraction buffer (25

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mM phosphate-buffered saline (PBS) and 100 mM NaCl, pH 7), blended, then sonicated for 10

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min in an ice bath using a Misonix Sonicator 3000 with micro-tip at 30% magnitude, followed by

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centrifugation at 3220 relative centrifugal force (RCF) for 10 min. The supernatant was then

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collected for further usage. Unused supernatant was frozen at -80 °C.

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D2D SDS-PAGE. Protein extraction samples were further diluted by extraction buffer 3

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times and incubated for 5 min in SDS sample buffer (pH 6.8) to a final concentration of 45 mM

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TrisHCl/1% SDS/10% glycerol/0.01% bromophenol blue). The SDS sample buffer does not

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contain disulfide reducing agent because some proteins may require disulfide bond(s) for kinetic

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stability. A 100 µl aliquot of the lysate solution was loaded without prior heating onto a well of a

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12% acrylamide gel (16 cm x 14 cm x 1.5 mm). Electrophoresis was performed in a Protean II xi

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cell (Bio-Rad, Hercules, CA) and run at 50 mA. The gel was kept at 10°C by using a circulating

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water bath. Running buffer contained 25 mM Tris base, 0.2 M glycine, and 0.1% SDS. After the

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first dimension run was completed (Fig. 2B), the gel strip was cut out and incubated for 10 min

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in equilibration buffer (50 mM TrisHCl/1% SDS/15% glycerol/0.02% bromophenol blue, pH 6.8)

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at 100°C (Fig. 2A). The gel strip was rinsed briefly and placed on top of a 12 cm x 14 cm x 2

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mm 12% acrylamide gel (Fig. 2B). A small amount of 12% acrylamide solution was used to fuse

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the strip to the stacking gel. The second-dimension separation was performed under similar



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conditions as the first-dimension run, except 65mA was used for each gel (Fig. 2A). Gels were

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stained with using Coomassie (Bio-Rad Biosafe), and the protein spots below the diagonal

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protein band were picked with a One Touch 2D gel spot picker (1.5 mm).

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Protein Digestion. Trypsin digestion of spots was performed using Trypsin Gold from

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Promega and followed product standard protocol. For key steps, spots were destained by 100

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mM NH4HCO3/50% acetonitrile (ACN), and then dehydrated in 100% ACN. Dithiothreitol (DTT)

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was used to break disulfide bonds and free cysteines were then recapped with iodoacetamide

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(IAA). Trypsin was diluted into 40 mM NH4HCO3/10% ACN to 20 µg/ml and spots were

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incubated overnight at 37°C.

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Liquid Chromatography–Mass Spectrometry. Peptide fractions were analyzed using an

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Agilent 1100-Series LC system coupled to an LTQ-Orbitrap mass spectrometer (Thermo

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Scientific, Bremen, Germany). The LC system was equipped with a 75 µm ID, 15 µm tip, and

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105 mm picochip (New Objective, Cambridge, MA) bed packed with 5µm BioBasic, (Thermo

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Scientific, Bremen, Germany). C18, 300A resin. Elution was achieved with a gradient of 0–5%

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buffer B (98% acetonitrile (ACN) in 1% formic acid) in 0.1 min, 5–90% B in 107 min and 90–100%

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B in 15 min. The flow rate was passively split from 0.35 mL/min to 50 nL/min. Nanospray was

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achieved using biased to 1.7 kV. The mass spectrometer was operated in data-dependent

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mode to switch between MS and MS/MS. The five most intense ions were selected for

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fragmentation in the linear ion trap using collisionally-induced dissociation at a target value of

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30,000.

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Database Search and Validation. Spectra were processed with the Protein Discovery

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software to generate peak lists, which were then analyzed with Mascot search engine version

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2.3.02 (Matrix Science, London, UK) using a concatenated forward/reverse database of the

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NCBInr, setting carbamidomethyl (C) as fixed, and oxidation (M) and acetylation (protein N-term)

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as variable modifications. A maximum of two missed cleavages were allowed, peptide tolerance

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was set to 50 ppm. and MS/MS tolerance to 0.6 Da. Search results were filtered with


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Rockerbox30 to a false discovery rate (FDR) of 1% using the concatenated database decoy

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method.

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Image Quantification. Destained gels were imaged by a Biorad Gel Doc XR+ system and

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then analyzed by the ImageJ, which has a feature to subtract the gel background from the

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whole D2D gel. Any proteins below the diagonal main line were classified as KSPs. The density

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of the background is measured and the area and average density of KSPs or diagonal are

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measured respectively. The average density of KSPs or proteins in the diagonal was then

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normalized by subtracting the background density. The normalized density multiplied by area

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generated the whole protein amount for KSPs (A-KSPs) or diagonal (A-non-KSPs). Percentage

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of KSPs (KSPs %) was calculated accordingly as A-KSPs/ (A-KSPs+ A-non-KSPs).

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RESULTS

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D2D SDS-PAGE of Beans and Peas Shows Variable Abundance of KSPs. On the basis

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of our observed correlation between kinetic stability and a protein’s resistance to the detergent

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SDS,13,

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proteome-level identification of KSPs.15 In D2D SDS-PAGE (illustrated in Figure 2A) SDS-

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resistant proteins migrate below the gel diagonal, and the identity of these KSPs may be

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subsequently determined via proteomics analysis.15 We used D2D SDS-PAGE to analyze

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protein extracts from 7 beans, 4 peas, and 1 lentil. These legumes were selected to cover a

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broad range of common sources of protein nutrition, and were purchased from local food stores

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to study legumes representative of what people ingest. We also included different varieties of

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Phaseolus vulgaris, known as “common bean”, for their nutritional importance and recognized

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presence of antinutritional factors that interfere with protein digestion.2 The selected common

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beans include black bean, navy bean, small red bean and kidney bean, all belonging to

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Phaseolus vulgaris. Among the other legumes are Lima bean (Phaseolus lunatus), soybean

14

we developed a diagonal two-dimensional (D2D) SDS-PAGE method for the



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(Glycine max), black-eyed pea (Vigna unguiculata), pigeon pea (Cajanus cajan), split pea

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(Pisum sativum), mung bean (Vigna radiate), red lentil (Lens culinaris) and chickpea (Cicer

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arietinum).

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The D2D SDS-PAGE data show Phaseolus beans and soybean contain significant amounts

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of KSPs, whereas the peas, red lentil, and mung bean have low amounts of KSPs (Figure 2B).

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To identify the KSPs, the spots below the diagonal of the D2D gels were picked and processed

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for mass spectrometry (MS) analysis. The resulting MS data was analyzed using the Protein

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Discovery proteomics database to identify each protein with high degree of certainty (see

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MATERIALS AND METHODS). A list of the KSPs identified is shown in Table 1, but the

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complete data, including the identity of each gel spot are shown in the information (Table 1 and

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Figure 1 of Supporting Information) available online. It should be noted that for some beans, in

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particular soybean, some stable soluble protein complexes or aggregates were present and

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migrated at the far left of the gel along the first dimension gel interface between the stacking

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and resolving gels. These protein spots were also analyzed by MS. Most of the KSPs in beans

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fall into two categories: globulin-like storage proteins and biodefense proteins. The hyperstability

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of biodefense proteins is well documented and is an important property to accomplish its

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protective function. However, the biological significance for the divergent KS of storage proteins

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– usually the most abundant proteins in legumes – is not clear.

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The Abundance of Kinetically Stable Proteins in Legumes Correlates With Their

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Digestibility. It is well known the digestibility of legumes is related to the stability of their

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proteins.3 However, the biophysical basis of this relationship is not clear. Our D2D SDS-PAGE

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results (Figure 2B) suggest the abundance of KSPs is an important factor in determining legume

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digestibility. To probe this relationship, we used Image J to quantify the relative amount of KSPs

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in the gels of each legume studied, and plotted the data alongside the percentage of their

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protein non-digestibility obtained from published in vitro protein digestibility (IVPD) rates (Figure

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3).16-32 The general correlation seen in Figure 3 between the abundance of KSPs and in vitro


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digestibility data links these two properties in legumes, and implies that legume digestibility

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depends largely on the abundance of KSPs. Thus, common beans are generally more difficult to

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digest than peas, red lentil, and mung bean because the former contain greater amounts of

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KSPs. Our observation does not exclude other factors from playing a role in legume digestibility.

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For example, mung bean and red lentil have higher non-digestibility than may be expected from

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their low content of KSP (Figure 3). This may result from other antinutrition factors like the in

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vivo presence of protease inhibitors. Thus, although various factors contribute to in vivo protein

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digestion, our results obtained from the analysis of D2D SDS-PAGE data point to KS as an

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important factor.

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A Potential Link Between Kinetically Stable Proteins and Germination Temperature in

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Legumes. Climate plays a major role in the germination of all plants. In particular, legumes

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have maximum, minimum, and optimum germination temperatures, depending on their CO2

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fixation mechanism, and are classified as warm season or cool season legumes.33, 34 Warm

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season legumes usually have minimum germination temperatures (MGT) in the 15-18 °C range,

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whereas cool season legumes have MGT in the 2-10 °C range.33,

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temperature of legumes is an important property that has major importance in agriculture.33, 34

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Since temperature affects the rate of protein digestion in legumes, the KS of storage and

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biodefense proteins may influence the minimum germination temperature of legumes.

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The germination

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Most beans included in our study fall within the warm season category, whereas peas and

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red lentil are cool season legumes. To test whether the abundance of KSPs in the legumes

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studied here correlates with their minimum germination temperature, we plotted the percentage

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of KSPs (data from Figure 2B) against the MGT, as reported in previous studies.2, 33, 35-41 As

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shown in Figure 4, warm season beans with the highest percentage of KSPs (60-70%) also

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have the highest MGT (15-18 °C). In contrast, cool season legumes such as chickpea, red lentil

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and split pea have the lowest percentage of KSP (< 20%) and MGT (2-10 °C). Interestingly,



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mung bean, which is a highly digestible warm season legume with among the lowest amounts of

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KSPs, has a MGT that is similar to that of other warm season beans containing high levels of

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KSPs. To a lesser extent than mung bean, black-eyed pea and chickpea have relatively low

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percentage of KSP (15-22%) but their MGT (10 °C) is near the middle of the range. In summary,

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although beans with high levels of KSPs show the highest MGT, legumes with low amounts of

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KSPs exhibit low, medium, or high MGT. Thus, high abundance of hyperstable storage proteins

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in beans appears to favor higher MGT.

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DISCUSSION

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Protein Kinetic Stability is a Main Contributor to the Digestibility of Proteins in Beans and

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Peas. The use of D2D SDS-PAGE for determining the proteomics-level KSPs in legumes or

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plants is unprecedented. The results of this study show a strong novel correlation between the

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abundance of KSPs and the non-digestibility of proteins in beans and peas (Figure 3). It has

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been known for decades that the “stability” of storage proteins in legumes represents an

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antinutritional factor limiting their digestibility and nutritional benefit.1 However, prior to our work

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the type of protein stability responsible for legume digestibility had not been determined. The

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term “protein stability” is typically used in reference to thermodynamic/equilibrium stability. But, it

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is known that kinetic stability, not thermodynamic stability, is the main determinant of protein

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susceptibility to proteolytic degradation (Figure 1).11 Because D2D SDS-PAGE identifies

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proteins that are SDS-resistant,15 a property that arises from a protein’s high kinetic stability,13, 14

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our results provide a biophysical understanding of the specific type of stability (i.e. kinetic

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stability) in legume proteins that largely determine their proteolytic susceptibility.

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Although the structural features of proteins that determine their kinetic stability remain poorly

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understood, topological complexity and flexibility appear to be important factors.42 In the case of

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the legume KSPs found here, it is known that the globulin storage proteins, phaseolin, vicilin,


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and beta-conglycinin have similar predicted structures, but exhibit significant differences in their

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proteolytic susceptibility that correlate with their different flexibility in solution.43 However, only

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the 3D structure of phaseolin (PDB code: 2PHL) from small red bean has been solved, limiting

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our ability to gain new insight about the structural basis of KS (or lack thereof) of these legume

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storage proteins. 2PHL phaseolin is a trimer consisting of identical subunits of 397 amino acids

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(Figure 6). Each monomer is composed of two “Jelly roll” beta-barrel structures. It also contains

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a linker region between the two barrel structures, and the initial stage of germination has been

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mapped to proteolytic cleavage at the end (res 220-221, labeled red in Fig. 6) of this linker.44 It

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is known this barrel structure is more rigid in common beans and more flexible in peas,

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consistent with the high KS of storage proteins in common bean.43 Interestingly, the structural

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features of this phaseolin is consistent with previous studies, where we have observed that

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oligomeric and beta-rich proteins appear more likely to possess kinetic stability than monomeric

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alpha-helix-rich proteins.13, 15 As the structure of more phaseolin and other storage proteins are

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solved, it will allow further studies to understand the structural basis of their KS and how it is

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modulated by other proteins and environmental factors to regulate its digestibility.

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The unique capability of D2D SDS-PAGE for identifying KSPs at a proteomics level may be

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used to discover KSPs in other legumes, including those in the wild with unique resilience. By

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identifying KS as a key factor limiting protein digestibility in legumes, our results identify a

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fundamental biophysical property that could be rationally targeted in the design of more

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digestible storage proteins. In addition, our approach of identifying KSPs in legumes could

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complement traditional in vitro digestibility assays for potentially predicting the nutritional

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prospects of genetically modified legumes, as well as for fundamental studies aimed towards

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understanding the biological roles of KSPs in legumes.

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Potential Role of Protein Kinetic Stability in Biodefense and Timing the Germination

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of Legumes. The potential biological roles of KSPs in legumes are intriguing. As showed in



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Figure 4, cool season legumes, which germinate at lower temperature, have less abundance of

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KSPs than most warm season legumes. This relationship is unlikely to be coincidental and may

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play a role in seed germination. Although other factors, including location of proteolytic sites and

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protease content and function play important roles in determining the efficiency of storage

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protein degradation during germination, KS may also be a significant factor. Since storage

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proteins are a source of nutrients during seed germination, the timing and kinetics of their

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degradation is important. The KS of storage proteins may place constraints on the

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environmental conditions required for their digestibility. For example, it is well known that

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temperature plays an important role in legume germination,

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KS (i.e. the degradability) of storage proteins together with the activity of endogenous proteases,

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may contribute to determining germination temperatures. This is consistent with the observation

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that legumain-like proteinase (LLP), the enzyme responsible for cleaving the phaseolin

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polypeptide chain, works efficiently at 30 °C, but is less efficient at lower and higher

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temperatures.45 This proposed relationship between the abundance of kinetically stable storage

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proteins and protease activity in warm-season and cool-season legumes is illustrated in Figure

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5. At low temperatures (i.e. ≤ 10 °C) the proteolytic activity may be sufficient to degrade the

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storage proteins in cool-season legumes, leading to germination. In contrast, for warm-season

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legumes, the proteolytic activity at ≤ 10 °C may not be sufficient to degrade the kinetically stable

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storage proteins, resulting in no germination. At higher temperatures (i.e. ≥ 15 °C) both warm-

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season and cool-season may germinate because the proteolytic activity is sufficient to degrade

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even the KSPs, which may also be conformationally more susceptible to proteolysis at higher

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temperatures. It should be noted the diagram in Figure 5 is just meant to illustrate how the

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potential relationship among temperature, proteolytic efficiency, and the KS of storage proteins

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may affect the MGT of legumes.

33, 34

and therefore it is plausible the

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While most of the KSPs we identified were storage proteins, some were biodefense proteins

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(Table 1). The resilience of such biodefense proteins must be essential for them to function


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effectively at warmer temperatures when biotic stress is higher. Interestingly, kinetically stable

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arcelin and alpha-amylase inhibitor were found abundantly in Lima bean but not in other

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legumes tested in this study. These lectin seed proteins have biodefense functions, conferring

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resistance against insects.46, 47 Arcelin binds the chitin in the digestive system of insects, but is

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not harmful to higher animals. Also, due to their high abundance in Lima beans, they also serve

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as storage proteins. Amylase activity levels are known to decide the dormancy break level in

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plants. Amylase inhibitor will keep the amylase activity level low to maintain dormancy. Amylase

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inhibitor is highly abundant and kinetically stable in Lima bean, which may be a factor explaining

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why Lima bean needs high soil temperatures of 45-55 °C to break dormancy.48 For comparison,

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common beans only need 16-35 °C to break the dormancy.34 Furthermore, in contrast to

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cultivated seed, wild seeds need higher temperature to break dormancy, suggesting these may

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have more dormancy-related KSPs.49 In general, the higher presence of biodefense KSPs in

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warm-season legumes is consistent with the need for such protection during the warmer climate,

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when biotic stress is relatively high. Cool-season legumes may not have experienced the

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selective pressure for developing biodefense proteins with high KS, but rather for developing

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easier to degrade storage proteins to allow germination at lower temperatures. Overall, the KS

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or lack thereof of storage and biodefense proteins may work synergistically along with other

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factors for timing legume germination and dealing with environmental stress.

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Kinetically Stable Proteins in Beans and Peas: Agricultural Implications. There is much

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interest in enhancing the properties of legume crops for improved sustainability. To what extent

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the KS of legume proteins may be exploited to help accomplish these goals is not yet clear, but

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some possibilities emerge from our study. First, the incorporation of newly discovered or

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currently known kinetically stable biodefense proteins could enhance ongoing efforts to reduce

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the need for pesticides and/or increase the viability of crops. Kinetic Stability would allow

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resistance against the digestive system of insects or bacteria to maintain its biodefense function.

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An interesting example is arcelin, which has not been found in cultivated common beans, but is



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present in wild common ancestor species.49 Some wild common beans contain arcelin, and it

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has been shown that a higher arcelin/phaseolin ratio correlates with greater insect resistance.46

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Alpha-amylase inhibitor has also been successfully transferred into pea to increase insect

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resistance to bruchid larvae.47 More such biodefense proteins must exist in nature. Thus, D2D

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SDS-PAGE may be used to screen wild legumes for the discovery of safe kinetically stable

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biodefense proteins that could be effectively used to create insect-resistant crops. A second

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area where the KS of legume proteins could be considered for enhancing crops relates to

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converting cool season into warm season legumes by changing their CO2 fixation pathways

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from C3 to C4. C4 crops have better yield and better water and nitrogen use efficiency. Among

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the potential advantages of having abundant KSPs in C4 crops, our results here suggest

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storage proteins with higher KS are less likely to be prematurely proteolyzed at lower

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temperatures. In addition, incorporating kinetically stable biodefense proteins should make

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crops more effective against the higher biotic stress of warm season temperatures. Furthermore,

328

engineering-in dormancy-related proteins with higher KS (e.g. alpha-amylase inhibitor in Lima

329

beans) would delay germination until a higher temperature is reached.

330

The presence of KSPs in legumes also has implications for farming and nutrition. The

331

digestibility of legume proteins is of major interest because of its nutritional impact in humans

332

and animals. It is also a major factor in the development of allergy.6 In common beans, the most

333

abundant proteins of the lectin family (arcelin, phytohaemagglutinin and alpha-amylase inhibitor)

334

are regarded as major antinutritional factors. Although legume protein digestibility is a concern

335

in human nutrition that is minimized by proper cooking, it is a major issue in farming animals.

336

Farm cattle are fed raw legumes, and therefore the presence of KSPs is likely to reduce the

337

protein utilization efficiency. This is consistent with known nutritional problems, including food

338

intolerance, associated with legume diet in young cattle.8 Of particular concern is soybean,

339

which is the major legume source of animal feed and known to trigger symptoms due to food


Page 15 of 32


340

intolerance. Bean lines producing seeds without the lectins mentioned above were developed to

341

improve nutritional characteristics of bean seeds used for human consumption and potentially,

342

for animal feeding.50 Thus, KS, a property in proteins that nature selected for to adapt and

343

survive, is also a major reason for the reduced nutritive value of legume proteins. An ideal

344

solution to this conundrum would be engineering crop-enhancing KSPs into legumes that could

345

be easily converted into highly digestible protein after a specific treatment (e.g. heating). The

346

use of D2D SDS-PAGE for convenient assessment of KS at the level of plant, seed, and food

347

could move this challenging goal forward.

348 349

ASSOCIATED CONTENT

350

Supporting Information

351

The Supporting Information is available free of charge on the ACS Publications website.

352

Kinetically stable proteins identified via proteomics analysis (Table 1); Protein spots on D2D

353

SDS-PAGE data identified by proteomics analysis (Figure 1); 1D SDS-PAGE of unboiled and

354

boiled samples of several beans to estimate the MW of the various SDS-resistant proteins

355

(Figure 2).

356 357

AUTHOR INFORMATION

358

Corresponding Author

359

*Phone: 518-276-6969. Fax: 518-276-4887. E-mail: [email protected].

360

Funding

361

Research was funded by a grant from the US National Science Foundation (#1158375) to W.C.

362

Notes

363

The authors declare no competing financial interest.

364



365

ABBREVIATIONS USED

366

ACN, acetonitrile, D2D SDS-PAGE, diagonal two-dimensional sodium dodecyl sulfate

367

polyacrylamide gel electrophoresis; DTT, Dithiothreitol; IAA, iodoacetamide; IVPD, in vitro

368

protein digestibility; LLP, legumain-like proteinase; KS, kinetic stability; KSP, Kinetically stable

369

proteins; MS, mass spectrometry; MGT, minimum germination temperature.


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370

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371

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ultrasound and high hydrostatic pressure during soaking. Cereal Chem. 2007, 84, 518-521.

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affected by cooking time. J. Sci. Food Agr. 1999, 79, 2025-2028.



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of some legume flours. Food Chem. 2009, 114, 1063-1068.

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(32) Palander, S.; Laurinen, P.; Perttila, S.; Valaja, J.; Partanen, K., Protein and amino acid

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digestibility and metabolizable energy value of pea (Pisum sativum), faba bean (Vicia faba) and

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lupin (Lupinus angustifolius) seeds for turkeys of different age. Animal Feed Sci. Tech. 2006,

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(33) Butler, T. J.; Celen, A. E.; Webb, S. L.; Krstic, D.; Interrante, S. M., Temperature affects the

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germination of forage legume seeds. Crop Sci. 2015, 54, 2846-2853.

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Scien. Agron. 2006, 28, 155-164.

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(35) Simon, E. W.; Minchin, A.; McMenamin, M. M., The Low Temperature Limit For Seed

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Germination. New Phytol. 1976, 77, 301-311.

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(36) Krauss, F. J., The pigeon pea (Cajanus indicus)-its improvement, culture, and utilization in

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Hawaii. Hawaii Agric. Exp. Sta. Bull. 1932, 64.

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(37) Ismail, A. M.; Hall, A. E.; Close, T. J., Allelic variation of a dehydrin gene cosegregates with

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chilling tolerance during seedling emergence. Proc. Natl. Acad. Sci. 1999, 96, 13566-13570.

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(38) Wolk, W. D.; Herner, R. C., Chilling injury of germinating seeds and seedlings. Hortscience

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1982, 17, 169-173.

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(39) Ismail, A. M.; Hall, A. E.; Close, T. J., Chilling tolerance during emergence of cowpea

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associated with a dehydrin and slow electrolyte leakage. Crop Sci. 1997, 37, 1270-1277.

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(40) Ellis, R. H.; Covell, S.; Roberts, E. H.; Summerfield, R. J., The Influence of Temperature on

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Seed-Germination Rate in Grain Legumes .2. Intraspecific Variation in Chickpea (Cicer-

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Arietinum-L) at Constant Temperatures. J. Exper. Bot. 1986, 37, 1503-1515.

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(41) Ellis, R. H.; Barrett, S., Alternating Temperatures and Rate of Seed-Germination in Lentil.

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Ann Bot-London 1994, 74, 519-524.


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(42) Broom, A.; Ma, S. M.; Xia, K.; Rafalia, H.; Trainor, K.; Colon, W.; Gosavi, S.; Meiering, E.

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M., Designed protein reveals structural determinants of extreme kinetic stability. Proc. Natl.

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Acad. Sci. 2015, 112, 14605-14610.

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(43) Deshpande, S. S.; Damodaran, S., Conformational characteristics of legume 7S globulins

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as revealed by circular dichroic, derivative u.v. absorption and fluorescence techniques. Int. J.

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Pept. Protein Res. 1990, 35, 25-34.

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(44) Seo, S. B.; Tan-Wilson, A.; Wilson, K. A., Protease C2, a cysteine endopeptidase involved

478

in the continuing mobilization of soybean beta-conglycinin seed proteins. Bba-Protein Struct M

479

2001, 1545, 192-206.

480

(45) Rotari, V.; Senyuk, V.; Horstmann, C.; Jivotovskaya, A.; Vaintraub, I., Proteinase A-like

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enzyme from germinated kidney bean seeds. Its action on phaseolin and vicilin. Physiol. Plant.

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1997, 100, 171-177.

483

(46) Cardona, C.; Kornegay, J.; Posso, C. E.; Morales, F.; Ramirez, H., Comparative value of

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four arcelin variants in the development of dry bean lines resistant to the Mexican bean weevil.

485

Entomol. Exp. Appl. 1990, 56, 197-206.

486

(47) Shade, R. E.; Schroeder, H. E.; Pueyo, J. J.; Tabe, L. M.; Murdock, L. L.; Higgins, T. J. V.;

487

Chrispeels, M. J., Transgenic Pea Seeds Expressing the α-Amylase Inhibitor of the Common

488

Bean are Resistant to Bruchid Beetles. Nat. Biotechnol. 1994, 12, 793-796.

489

(48) Degreef, J.; Rocha, O. J.; Vanderborght, T.; Baudoin, J. P., Soil seed bank and seed

490

dormancy in wild populations of lima bean (Fabaceae): Considerations for in situ and ex situ

491

conservation. Amer. J. Bot. 2002, 89, 1644-1650.

492

(49) Pena-Valdiva, C., Breaking dormancy of wild common bean Phaseolus vulgaris L. with high

493

temperatures. Annual Report 2000, 43, 204-205.

494

(50) Campion, B.; Perrone, D.; Galasso, I.; Bollini, R., Common bean (Phaseolus vulgaris L.)

495

lines devoid of major lectin proteins. Plant Breeding 2009, 128, 199-204.



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496

FIGURED LEGENDS

497

Figure 1. Free energy diagrams to illustrate the different types of protein stability and their

498

impact on proteolytic susceptibility. (A) Kinetically stable proteins (KSPs) are characterized by

499

having a high activation energy (∆G≠) for unfolding that virtually traps them in their native state

500

(N). The stability of KSPs is under kinetic control because their unfolding rate is very slow,

501

resulting in resistance to proteolysis, and thereby a longer half-life. (B) The thermodynamic

502

stability (TS) of proteins is defined as the difference in free energy (∆GU) between the native (N)

503

and unfolded (U) states. Since most soluble proteins in nature have low activation energy for

504

unfolding, their N and U states exist in equilibrium, which favors the more stable N state. The

505

stability of such proteins is under thermodynamic control and their transient sampling of

506

unfolded states give rise to their susceptibility to proteolysis. (C) Proteins with very high

507

thermodynamic stability (i.e. large ∆GU) will often be kinetically stable because the lower free

508

energy of the native state may also result in a higher energy barrier for unfolding.

509 510

Figure 2. Determining the KSPs in legumes using D2D SDS-PAGE. (A) Diagram illustrating

511

how D2D SDS-PAGE uses SDS-resistance to separate KSPs (orange colored bands/spots)

512

from non-KSPs in soluble protein extracts. Unheated protein samples are first analyzed by SDS-

513

PAGE, and the relevant gel strip is then excised and heated in a boiling buffer solution

514

containing SDS. The heated gel strip is then analyzed by 2nd dimension SDS-PAGE. The

515

resulting 2D gel shows a diagonal pattern from the SDS-sensitive proteins migrating the same

516

distance in both dimensions of SDS-PAGE. However, SDS-resistant proteins (i.e. KSPs)

517

migrate less in the 1st dimension, and therefore migrate left of the gel diagonal. (B) D2D SDS-

518

PAGE results of protein samples extracted from 12 different legumes. See Methods for

519

experimental details. The spots left of the diagonal represent the KSPs present in the respective

520

legumes. Horizontal smearing of spots is caused by partial SDS binding to the protein in the 1st


Page 23 of 32


521

dimension run, indicating a diminished kinetic stability (e.g. kidney bean). In contrast, round

522

focused spots (e.g. Lima bean) identify proteins with higher kinetic stability. The spots/smeared

523

bands seen above the gel diagonal are the result of disulfide cross-linking caused by the

524

heating step prior to the 2nd dimension run.

525 526

Figure 3. Comparison between the abundance of KSPs and protein digestibility in different

527

legumes. The abundance of KSPs present in the legumes analyzed by D2D SDS-PAGE relative

528

to the total protein content was determined using Image J (see Methods) and plotted as the

529

percentage (%) of KSPs (red columns). The blue columns represent the extent of raw legume

530

resistance to digestibility obtained from published results of in vitro protein digestibility studies16-

531

32

532

standard deviation was obtained from the average of data from at least three experiments (red)

533

or publications (blue).

. The data was plotted as % non-digestibility for better comparison with % KSPs data. The

534 535

Figure 4. Comparison between the abundance of KSPs and the minimum germination

536

temperature of different beans/peas/lentil. Red symbols represent the percentage of KSPs

537

present in the legumes analyzed by D2D SDS-PAGE relative to the total protein content. Green

538

circles indicate the minimum germination temperature (right y-axis) reported for different

539

legumes2, 36-41. The white and gray backgrounds represent the warm season and cold season

540

legumes, respectively. The standard deviation was obtained from the average of data from at

541

least three experiments.

542 543

Figure 5. Diagram illustrating the potential relationship among temperature, kinetic stability of

544

storage proteins, and protease activity. This diagram attempts to present a scenario through

545

which the abundance of kinetically stable proteins may partially affect the germination

546

temperature. At low temperatures (i.e. ≤ 10 °C) the proteolytic activity must be sufficient to



Page 24 of 32

547

degrade the less stable storage proteins in cool-season legumes, leading to germination (top

548

scenario). In contrast, for warm-season legumes the proteolytic activity at low temperature is not

549

sufficient to degrade the kinetically stable storage proteins, resulting in no germination (middle

550

scenario). At higher temperatures (i.e. ≥ 15 °C) both warm-season and cool-season may

551

germinate because in both cases the proteolytic activity is sufficient to degrade even the KSPs.

552 553

Figure 6. Three-dimensional structure of phaseolin (2PHL) from Small red bean. The protein

554

consists of a trimer formed by the end-to-end association of identical monomers. The monomer

555

is 397 residues in length and folds into two beta barrel domains connected by a flexible link

556

region, which was not solved in the 3D structure. Proteolytic cleavage at the end (red arrow, res

557

220-221) of this linker region is known to be a key early event in the process of bean seed

558

germination.


Page 25 of 32


Table 1. Kinetically Stable Proteins Identified and their Properties Name Legume species MW (kDa)# PDB* Function/EC Phaseolin(alpha, beta type)

Phaseolus vulgaris (common bean)

48,49

2PHL

7S storage protein

Phytohemagglutinin

Phaseolus vulgaris (common bean)

30

1G8W

antibiosis lectin/biodefense

Phaseolin**(PHSL1, PHSL2,PHSL3)

Phaseolus lunatus (lima bean)

35**,25**,21**

2PHL

7S storage protein

Lectin 1 (Arcelin- like protein)


29

1AVB

storage protein and biodefense protein

Alpha-amylase inhibitor


28

1AVB

inhibits alphaamylase activity/biodefense

Trypsin inhibitor

Glycine max (soybean)

24

1BA7

inhibits trypsin/biodefense

Beta-Conglycinin (alpha chain, beta chain)


51,70~74

1ULK

7S storage protein

Glycinin(G1,G2,G3,G4)


56,54,54,64

1FXZ

11S storage protein

Lectin soybean agglutinin (SBA)


31

1G9F

binds to and toxic to epithelial cells

Vicilin

V. unguiculata (black eye pea) Cicer arietinum (chick pea)

50/51

2CV6

storage protein

Convicilin

L. culinaris (Red lentil) Pisum sativum (Split pea)

60/67

1IPJ

storage protein immunologically similar to vicilin

Legumin

Pisum sativum (Garden pea) Cicer arietinum (chick pea)

59/56

3KSC

11S storage protein

#

Some proteins might be located higher in the gel (Figure 2 and Supporting Information Figure 2) than expected because the MW in this table (except for Lima bean) only includes the polypeptide chain, not the glycan. *Nearest homology Protein Data Bank (PDB) structures are found with high percentage coverage rate (80%) and more than 50% positive rate. ** When Lima bean matures, endoproteolytic cleavage of the precursor glycosylated phaseolin generates 3 stable fragments. PHSL1 (~35 kDa, the C terminal fragment) and PHSL2 (~25 kDa, the N-terminal fragment) are glycosylated, whereas PHSL3 (~21 kDa) is the unglycosylated form of PHSL2.



TOC Figure 83x44mm (300 x 300 DPI)


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190x254mm (300 x 300 DPI)



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190x254mm (300 x 300 DPI)



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Kinetic Stability of Proteins in Beans and Peas ... - ACS Publications

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