# Rational engineering of improved protein crystallization

> **NIH NIH R01** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2021 · $319,950

## Abstract

This grant application addresses the major obstacle to using crystallographic methods to gain insight into
biological function, which is the failure of most naturally occurring proteins to yield crystals suitable for x-ray
structure determination. The goal of the current project is to develop methods for rational engineering of the
sequence of a protein to produce high quality crystals. Structural genomics consortia systematically confirmed
that crystallization is the major obstacle to determining the atomic structure of proteins using x-ray diffraction
methods. Previously published work from the Hunt laboratory employed computational analysis of large-scale
crystallization trials to demonstrate that protein surface properties, particularly the mean entropy of exposed
sidechains, are a dominant determinant of crystallization propensity. This study identified a variety of sequence
properties that correlate with crystallization success, including the content of several individual amino acids.
However, every one of the amino acids that positively correlates with crystallization success negatively
correlates with protein solubility, and vice-versa. This effect severely limits the efficacy of using single amino
acid substitutions to engineer improved protein crystallization properties because crystallization probability is
low unless the initial protein preparation is monodisperse and soluble. In this application, we propose to use a
suite of computational methods to identify more complex sequence epitopes that promote successful protein
crystallization without impairing solubility. Computational analyses will be used to select sites for introduction of
such epitopes in a manner likely to preserve protein function and stability. These novel crystallization-
engineering methods will be critically evaluated and optimized using studies in which the thermodynamic
stability, solubility, and crystallization properties of purified mutant proteins are determined experimentally. Our
preliminary data for these proposed studies support the efficacy of the approach while also showing that the
crystallization propensity of a protein is not directly coupled to its thermodynamic solubility. Therefore, if the
underlying stereochemical and thermodynamics mechanisms were sufficiently well understood, then it should
be possible to engineer improved protein solubility in parallel with improved crystallization propensity. The twin
objectives of the research proposed in this grant application are to elucidate these mechanisms while also
generating rigorously validated methods for improving protein crystallization and solubility. Successful
development of efficient methods for engineering improved protein crystallization would facilitate a wide variety
of structural/functional biology projects, including projects focused on drug-discovery and structural
characterization of macromolecular complexes.

## Key facts

- **NIH application ID:** 10249105
- **Project number:** 5R01GM127883-04
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** JOHN Francis HUNT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $319,950
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10249105

## Citation

> US National Institutes of Health, RePORTER application 10249105, Rational engineering of improved protein crystallization (5R01GM127883-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10249105. Licensed CC0.

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